Cross-reacting monoclonal antibodies specific for E-selectin and P-selectin

ABSTRACT

This invention provides monoclonal antibodies that bind to both E-selectin and to P-selectin, and inhibit the binding of these proteins to counterreceptors. The invention also provides nucleic acids encoding these antibodies and methods for using the antibodies in the treatment of inflammatory conditions.

This application is the United States national stage of PCT/US95/07302,filed Jun. 7, 1995, which is a continuation-in-part of application Ser.No. 08/259,963, filed Jun. 14, 1994, now U.S. Pat. No. 5,622,701.

BACKGROUND OF THE INVENTION

The ability of cells to adhere to one another plays a critical role indevelopment, normal physiology, and disease processes such asinflammation. This ability is mediated by adhesion molecules, generallyglycoproteins, expressed on cell membranes. Often, an adhesion moleculeon one cell type will bind to another adhesion molecule expressed on adifferent cell type, forming a receptor counter-receptor pair. Threeimportant classes of adhesion molecules are the integrins, selecting,and immunoglobulin (Ig) superfamily members (see Springer, Nature346:425 (1990); Osborn, Cell 62:3 (1990); Hynes, Cell 69:11 (1992).These molecules are vital to the interaction of leukocytes and plateletswith themselves and with the extracellular matrix and vascularendothelium.

The selectin family of receptors are so named because of theirlectin-like domain and the selective nature of their adhesive functions.There are three known selecting, L-selectin (also known as LECAM-1,Mel-14 or LAM-1 or CD62L), E-selectin (also called ELAM-1 or CD62E) andP-selectin (also known as CD62, CD62P, GMP140 or PADGEM). The selectingare highly homologous, containing a 120 amino acid (aa) N-terminallectin domain, an EGF-like domain, a variable number of multiple shortconsensus repeat (SCR) domains homologous to those found in complementregulatory proteins, followed by a transmembrane domain and shortcytoplasmic tail. See Siegelman et al., Science 243:1165-1172 (1989);Lasky et al., Cell 56:1045-1055 (1989); Tedder et al., J. Exp. Med.170:123-133 (1989); Johnson et al., Cell 56:1033-1044 (1989); Bevilacquaet al., Proc. Natl. Acad. Sci. USA 84:9238-9242 (1987), Bevilacqua etal., Science 243:1160-1165 (1989), Bevilacqua et al., J. Clin. Invest.91:379-387 (1993), Camerini et al., Nature 280:496-498 (1989). Theselecting have overlapping but distinct specificities forcounterreceptors. See Bevilacqua et al., J. Clin. Invest. 91:379-387(1993); Feize, Current Opinion in Struct. Biol. 3:701-710 (1993); Berget al., Biochem. Biophys. Res. Comm. 184:1048-1055 (1992); Foxall etal., J. Cell Biol. 117:895-902 (1992); Larsen et al., J. Biol. Chem.267:11104-11110 (1992); Polley et al., Proc. Natl. Acad. Sci. USA88:6224-6228 (1991) (each of which is incorporated by reference in itsentirety for all purposes).

P-selectin is constitutively expressed by both platelets and endothelialcells where it is stored in α-granules or Weibel-Palade bodies for rapid(seconds to minutes) translocation to the cell surface upon activationby, for example, thrombin or histamine (McEver et al., J. Biol. Chem.250:9799-9804 (1984); Hsu-Lin et al., J. Biol. Chem. 264:8121-9126(1984)). E-selectin is expressed by activated endothelial cells (e.g.,after TNF-α or IL-1 stimulation for 6-8 hr). Its expression iscontrolled at the transcriptional level (Bevilacqua et al., 1987, supra;Bevilacqua et al., 1989, supra). P-selectin and E-selectin both bind toneutrophils and monocytes (Larsen et al., Cell 59:305-312 (1989);Johnston et al., Cell 56:1033-1044 (1989); Bevilacqua et al., 1987,supra; Bevilacqua et al., 1989, supra), as well as subsets oflymphocytes (Picker et al., Nature 349:796-799 (1991); Shimizu et al.,Nature 349:799-802 (1991); Moore et al., BBRC 186:173-181 (1992)).L-selectin is constitutively expressed by leukocytes, and mediateslymphocyte adhesion to peripheral lymph node high endothelial venules(HEV) (Gallatin et al., Nature 304:30-34 (1983); Berg et al., Immunol.Rev. 108:5-18 (1989); Berg et al., J. Cell. Biol. 114:343-349 (1991)),and neutrophil adhesion to cytokine-activated endothelial cells (Hallmanet al., Biochem. Biophys. Res. Comm. 174:236-243 (1991); Smith et al.,J. Clin. Invest. 87:609-618 (1991); Spertini et al., J. Immunol.147:2565-2573 (1991)). L-selectin is a counter-receptor on neutrophilsfor both E-selectin and P-selectin (Kishimoto et al., Blood 78:805-811(1990), Picker et al., Cell 66:921 (1991)), although all three selectinsprobably have other counter-receptors as well.

E-selectin, P-selectin and L-selectin mediate leukocyte-endothelial celland platelet-leukocyte adhesive interactions during inflammation(Bevilacqua et al., 1993, supra). All three selectins have beendemonstrated to participate in an initial “rolling” interaction ofleukocytes with activated endothelium (von Andrian et al., Proc. Natl.Acad. Sci. USA 88:7538-7542 (1991); Ley et al., Blood 77:2553-2555(1991); Abassi et al., J. Clin. Invest. 92:2719-2730 (1993); Dore etal., Blood 82:1308-1316 (1993); Jones et al., Biophys. J. 65:1560-1569(1993); Mayadas et al., Cell 74:541-554 (1993)). This initialinteraction precedes CD18-integrin-mediated adhesion and subsequentmigration of neutrophils through the endothelium and into inflamedtissue sites (Lawrence et al., Cell 65:859-873 (1991); von Andrian etal., Am. J. Physiol. 263:H1034-H1044 (1992)). Depending on the nature ofinflammatory stimuli and time after initiation of inflammatory response,either E-selectin or P-selectin may be functionally dominant inpromoting neutrophil-mediated tissue damage.

In principle, antibodies or other antagonists of the selecting couldabort the adhesion process, thereby preventing neutrophils from bindingto the endothelium and from extravasating into tissues. A substantialnumber of antibodies specific for one of the selecting have beenreported. Some of these antibodies have been reported to block bindingof selecting to counterreceptors in vitro. Some of the antibodies havealso been reported to block selectin-mediated interactions in animalmodels in vivo. For example, antibodies to E-selectin have been reportedto protect against neutrophil-mediated damage in an IgG complex model oflung injury in the rat (Mulligan et al., J. Clin. Invest. 88:1396(1991)). Antibodies to P-selectin have been reported to protect againstacute lung injury induced by intravenous injection of cobra venom factor(Mulligan et al., J. Clin. Invest. 90:1600-1607 (1992)), as well as in arat model of systemic endotoxemia (Coughlan et al., J. Exp. Med.179:329-334 (1994)). Antibodies to P-selectin have also been reported tobe protective in a cat model of myocardial ischemia and reperfusioninjury (Weyrich et al., FASEB J. 7:A785 (1993)).

Although some antibodies against E-selectin and P-selectin have shownblocking activity, many, if not most, antibodies specific for E-selectinor P-selectin are nonblocking (see, e.g., Bevilacqua et al., 1989,supra; Erbe et al., J. Cell Biol. 119:215-227 (1992)). That is, theseantibodies bind to epitopes in the extracellular domains of E-selectinor P-selectin that do not directly participate in counterreceptorbinding or the subsequent cellular adhesion process. The prevalence ofnonblocking antibodies suggests that only small regions of theextracellular domain participate directly in binding or influencebinding. Thus, de novo screening of antibodies generated againstE-selectin or P-selectin would be expected to generate mainlynonblocking antibodies.

Despite the large number of antibodies isolated to-date against thethree selectins, there have been few reports of crossreacting antibodiesthat bind to more than one selectin. Crossreacting antibodies might becapable of aborting the inflammatory process at more than one level,thereby providing more broadly useful therapeutic agents forneutrophil-mediated inflammatory conditions than antibodies specific fora single selectin. One antibody has been reported to crossreact withhuman E-selectin and dog L-selectin but not with the two selectins fromthe same species (Abassi et al., J. Immunol. 147:2107-2115 (1991)). Asecond antibody has been reported to crossreact with human E-selectinand L-selectins (Jutila et al., J. Exp. Med. 175:1565-1573 (1992);WO/9324614). However, no antibody has been isolated that binds to bothP-selectin and E-selectin, much less blocks the functions of both ofthese molecules.

Accordingly, there is a need for antibodies that bind to both E-selectinand P-selectin, preferably so as to block the capacity of both of thesemolecules to participate in adhesion reactions with counterreceptors.The present invention fulfills this and other needs.

SUMMARY OF THE INVENTION

The invention provides monoclonal antibodies that have a binding sitethat specifically binds to P-selectin and to E-selectin. For many suchantibodies, specific binding of the antibody to the P-selectin inhibitsbinding of the P-selectin to a counterreceptor of P-selectin, andspecific binding of the antibody to E-selectin inhibits binding of theE-selectin to a counterreceptor of E-selectin. Counterreceptors ofE-selectin and P-selectin are expressed on the surface of cells such asHL-60 cells and neutrophils. Exemplary antibodies are designated 57C.29,2C9.11 and 1D8.10. Many of the antibodies of the invention compete withan exemplified antibody for specific binding to P-selectin and toE-selectin. Some antibodies of the invention also specifically bind toL-selectin, whereas others do not. In one embodiment the antibodyrecognizes an epitope of E-selectin comprising amino acids Q₂₁, R₂₂,Y₂₃, T₁₁₉, and A₁₂₀. In another embodiment, the antibodies bind to thesame epitope of E-selectin and/or P-selectin as antibody 5C7.29. Inaddition to intact antibodies, the invention also provides bindingfragments such as Fab, Fab′, F(ab′)₂, Fv or single-chain antibodies.

Some of the antibodies of the invention are non-human, e.g., mouse,whereas others are humanized or human antibodies. A humanized antibodycomprises a humanized heavy chain variable region and a humanized lightchain variable region. The humanized light chain variable region cancomprise complementarity determining regions (e.g., CDR1, CDR2, CDR3)having amino acid sequences from the light chain of a mouse, antibodyselected from the group consisting of 5C7.29, 2C9.11 and 1D8.10, andhaving a variable region framework sequence substantially identical to ahuman light chain variable region framework sequence. The humanizedheavy chain variable region can comprise complementarity determiningregions (e.g., CDR1, CDR2 and CDR3) having amino acid sequences from thecorresponding mouse antibody heavy chain, and having a variable regionframework sequence substantially identical to a human heavy chainvariable region framework sequence. The antibodies optionally containconstant regions substantially identical to human constant regions.

In particular embodiments of the humanized antibodies of this invention,the humanized light chain variable region has a sequence substantiallyidentical to the mature sequence depicted in FIG. 8A [SEQ ID NO:6] andthe humanized heavy chain variable region has a sequence substantiallyidentical to the mature sequences depicted in FIG. 8B [SEQ ID NO:8].More particularly, this invention provides humanized antibodies wherein(a) the humanized light chain variable region has the sequence:X₁IX₂X₃TQSPSS LSASVGDRVT ITCSASSSX₁₁P YX₁₂HWYQQKPG KAPKLLIYDTSNX₁₃X₁₄X₁₅GVPX₄R X₇SGSGSGTX₅X₆ TX₈TISSLQPE DX₉ATYYCX₁₆X₁₇W SSDPFTFGX₁₀GTKVEIK [SEQ ID NO:9], wherein X₁=D or Q; X₂=Q or V; X₃=M or L; X₄=S orA; X₅=S or D; X₆=Y or F; X₇=F or I; X₈=L or F; X₉=F, I or A; X₁₀=Q, G orS; X₁₁=V, I or L; X₁₂=M or L; X₁₃=any amino acid; X₁₄=any amino acid;X₁₅=S or T; X₁₆=Q, N or H; and X₁₇=Q, N or H; and (b) the humanizedheavy chain variable region has the sequence: X₃VQLVESGGG LVQPGGSLRLSCAASGFTFS SFGX₇HWVRQA PGKGLEWVX₁F ISSGSSTIYY X₈X₉X₁₀X₁₁X₁₂X₁₃RFTISRDNX₄KNX₅LY LQMX₂SLRAED TAVYYCARPL PPFAYWGQGT LVTVSX₆ [SEQ ID NO:10];wherein, X₁=A or S; X₂=N or T; X₃=E, Q or D; X₄=S, A or P; X₅=T or S;X₆=A or S; X₇=M, I, V or L; X₈=any amino acid; X₉=any amino acid;X₁₀=any amino acid; X₁₁=V, A, I, L, M or F; X₁₂=R, K or Q; and X₁₃=G, A,D, T or S. In certain embodiments of the aforementioned antibodies, theCDR regions of the light and heavy chain variable regions have the sameamino acid sequence as the CDR sequences of FIGS. 8A and 8B. That is, inthe human light chain variable region, X₁₁=V; X₁₂=M; X₁₃=L; X₁₄=A;X₁₅=S; X₁₆=Q; and X₁₇=Q; and in the heavy chain variable region, X₇=M;X₈=A; X₉=D; X₁₀=T; X₁₁=V; X₁₂=R; and X₁₃=G. In another embodiment, thevariable light and heavy chain regions have the amino acid sequencedepicted in FIGS. 8A and 8B.

In another aspect, the invention provides purified nucleic acid segmentsencoding a light or heavy chain variable region of one of the monoclonalantibodies discussed above.

The invention also provides stable cell lines capable of producing theantibodies described above. The stable cell lines comprise nucleic acidsegments respectively encoding the heavy chain and light chain of anantibody described above. The segments are operably linked to first andsecond promoters to allow expression of the heavy and light chains.

The invention further provides pharmaceutical compositions comprisingthe antibodies described above and methods of treatment using the same.The methods of treatment are particularly effective for inflammatorydiseases including conditions such as ischemia-reperfusion injury, adultrespiratory distress syndrome, sepsis, psoriasis and autoimmune disease.

In another aspect, the invention provides methods of generating anantibody capable of blocking E-selectin and/or P-selectin mediatedfunctions. The method comprises concurrently or consecutively immunizinga mammal with P-selectin and E-selectin. B-cells from the mammal areimmortalized to generate immortalized cells producing antibodies. Animmortalized cell is selected producing an antibody that specificallybinds to E-selectin and to P-selectin.

The invention further provides methods of detecting E-selectin andP-selectin bearing cells in a biological sample suspected of containingthe cells. The method comprises contacting the sample with an antibodyas described above to form an immune complex with the E-selectin and/orP-selectin bearing cells. The presence of the immune complex is thendetected to indicate the presence of the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: Crossreacting antibody 5C7.29 binds to naturallyoccurring human E-selectin. (a) Binding of known anti-E-selectinantibody H18/7 to activated (black histograms) and resting (greyhistograms) HUVEC cells. (b) Binding of crossreacting antibody 5C7.29 toactivated and resting HUVEC cells. FACS fluorescence intensity isindicated by the X axis.

FIGS. 2A and 2B: Crossreacting antibody 5C7.29 binds to naturallyoccurring P-selectin. (a) Binding of known anti-P-selectin antibody WAPS12.2 to platelets detected by staining with secondary antibody (blackhistogram), compared with staining with secondary antibody alone(control, grey histogram). (b) Binding of 5C7.29 to platelets, shownsimilarly.

FIGS. 3A-B: Crossreactivity of 5C7.29 resides in a single monoclonalantibody. 5C7.29 antibody was incubated with excess of (a, c) parentL1-2 cells or (b, d) L1-2^(P-selectin) transfectants, and resultingsupernatants tested for reactivity with fresh samples ofL1-2^(P-selectin) (a, b) or L1-2^(E-selectin) cells (c, d) by FACSanalysis. These figures show that L1-2^(P-selectin) depletes reactivityfor E-selectin.

FIG. 4: Monoclonal antibody 5C7.29 blocks binding of HL-60(neutrophil-like) cells to TNF-α-activated HUVEC cells (expressingE-selectin). Average of four experiments.

FIG. 5. Monoclonal antibody 5C7.29 blocks binding of HL-60 cells toE-selectin transfectant cells. Average of four experiments.

FIG. 6. Monoclonal antibodies 5C7.29, 2C9.11 and 1D8.10 block binding ofplatelets to HL-60 cells as shown by platelet rosetting. The chart showsthe percentage of HL-60 cells with >2 platelets bound (rosetted).Average of three experiments.

FIGS. 7A-7B. Sequences of the CDNA (light chain—SEQ ID NO:1; heavychain—SEQ ID NO:3) and translated amino acid sequences (light chain—SEQID NO:2; heavy chain—SEQ ID NO:4) of the light chain (A) and heavy chain(B) variable regions of the mouse 5C7.29 antibody. The first amino acidof each mature chain is indicated by a double underline. The three CDRsin each chain are underlined.

FIGS. 8A-8B. Sequences of the synthetic DNA (light chain—SEQ ID NO.:5;heavy chain—SEQ ID NO:7) and translated amino acid sequences (lightchain—SEQ ID NO:6; heavy chain—SEQ ID NO:8) of the light chain (A) andheavy chain (B) variable regions of the humanized 5C7.29 antibody. Thefirst amino acid of each mature chain is indicated by a doubleunderline. The three CDRs in each chain are underlined.

FIG. 9. Schematic diagram of construction of humanized 5C7.29 antibodyvariable region genes.

FIG. 10. Humanized 5C7.29 antibody reactivity with E-selectin,P-selectin and L-selectin transfectants. L1-2 transfectant cell linesexpressing the indicated selectin were analyzed for reactivity withhumanized 5C7.29 by flow cytometry.

FIGS. 11A and 11B. Competitive binding of mouse and humanized 5C7.29antibodies to cells expressing E-selectin (A) or P-selectin (B).Increasing concentrations of cold competitor antibody were incubatedwith the cells in the presence of radiolabeled tracer mouse 5C7.29antibody, and the ratio of bound/free radioactivity was determined.

FIG. 12. Inhibition of HL-60 cell adhesion to CHO^(E-selectin) cells bymouse and humanized 5C7.29 antibodies. Fluorescently labelled HL-60cells were incubated with CHO^(E-selectin) cells in the presence of theantibodies at the indicated concentrations. After washing, adherentcells were counted microscopically. The results from a representativeexperiment performed with each sample in quadruplicate (+/−standarddeviation) are shown.

FIG. 13. Inhibition of platelet rosetting to HL-60 cells by mouse andhumanized 5C7.29 antibodies. Normal human platelets were incubated withHL-60 cells in the presence of the antibodies at the indicatedconcentrations. After fixation, the percent of HL-60 cells with greaterthan 2 platelets bound (rosetted) was determined. The results shown arefrom a representative experiment performed with each sample intriplicate (+/−standard deviation).

DEFINITIONS

The term “substantial identity” or “substantial homology” means that twopeptide sequences, when optimally aligned, such as by the programs GAPor BESTFIT using default gap weights, share at least 80 percent sequenceidentity, preferably 90 percent sequence identity, more preferably atleast 95 percent sequence identity or more (e.g., 99 percent sequenceidentity). Preferably, residue positions which are not identical differby conservative amino acid substitutions.

The term “substantially pure” or “isolated” means an object species isthe predominant species present (i.e., on a molar basis it is moreabundant than any other individual species in the composition), andpreferably a substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present. Generally, a substantiallypure composition will comprise more than about 80 to 90 percent byweight of all macromolecular species present in the composition. Mostpreferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

“Immunoglobulin,” “antibody” or “antibody peptide(s)” refers to anintact antibody, or a binding fragment thereof that competes with theintact antibody for specific binding. Binding fragments are produced byrecombinant DNA techniques, or by enzymatic or chemical cleavage ofintact immunoglobulins. Binding fragments include Fab, Fab′, F(ab′)₂, Fvand single-chain antibodies. An antibody other than a “bispecific” or“bifunctional” antibody is understood to have each of its binding sitesidentical.

An antibody substantially inhibits adhesion of a receptor to acounterreceptor when an excess of antibody reduces the quantity ofreceptor bound to counterreceptor by at least about 20%, 40%, 60% or80%, and more usually greater than about 85% (as measured in an in vitrocompetitive binding assay).

The term epitope includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics.

An antibody is said to specifically bind an antigen when thedissociation constant is≦1 μM, preferably≦100 nM and most preferably≦10nM.

The term patient includes human and veterinary subjects.

The term P-selectin counterreceptor denotes a protein other than anantibody that specifically binds to P-selectin at least in part bynoncovalent bonds. Specific binding maintains cells respectively bearingreceptor and counterreceptor in physical proximity and may alsotransduce a change in physical or functional phenotype in either of thecells or both. Other selectin counterreceptors are analogously defined.

DESCRIPTION OF THE PREFERRED EMBODIMENT I. Antibodies of the Invention

The invention provides antibodies that crossreact, i.e., specificallybind, with E-selectin and P-selectin. Preferred antibodies block thefunctions of both of these molecules.

A. General Characteristics of Antibodies

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989),Ch. 7 (incorporated by reference in its entirety for all purposes).

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame. The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat, Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987);Chothia et al., Nature 342:878-883 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methodsincluding fusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelnyet al., J. Immunol. 148, 1547-1553 (1992). Production of bispecificantibodies can be a relatively labor intensive process compared withproduction of conventional antibodies and yields and degree of purityare generally lower for bispecific antibodies. Bispecific antibodies donot exist in the form of fragments having a single binding site (e.g.,Fab, Fab′ and Fv).

B. Binding Specificity and Affinity

The immunoglobulins (or antibodies) of the invention exhibit specificbinding to both P-selectin and E-selectin. That is, a single bindingsite on an antibody has affinity for both P-selectin and E-selectin.Thus, the antibodies bind to epitopes that are common to both molecules.The antibodies bind to the natural and/or recombinant human forms forP-selectin and E-selectin (see Johnston et al., 1989, supra; Bevilacquaet al., 1989, supra). Some antibodies may also bind P-selectin and/orE-selectin from nonhuman species. Some of the antibodies alsospecifically bind to L-selectin (preferably human L-selectin (seeTedder, EPA 386,906 (1990)) whereas other antibodies of the invention donot. Surprisingly, the common epitopes bound by the crossreactingantibodies of the invention are also epitopes important for bothE-selectin and P-selectin to interact with their counterreceptors onactivated leukocytes, such as neutrophils. Thus, most crossreactingantibodies of the invention block the functional interactions ofE-selectin or P-selectin and usually those of both of these molecules.Some crossreacting antibodies also block the functional interactions ofL-selectin whereas others do not.

Blockage of P-selectin-mediated functions can be demonstrated in vitro.In vitro assays measure the capacity of an antibody to inhibit bindingof P-selectin to a counterreceptor. Suitable sources of P-selectin forsuch assays are purified P-selectin (or an extracellular domainthereof), cells transfected with P-selectin, activated endothelial cellsor platelets. Suitable sources of counterreceptor are leukocytes,neutrophils, monocytes, or HL-60 cells (ATCC CCL 240) and appropriatecell lines transfected with L-selectin. Neutrophils can be isolated fromwhole blood (preferably human blood) by Ficoll-Hypaque gradientcentrifugation. Neutrophils are usually pretreated with rabbit serum toblock Fc receptors before adding to a binding assay. When bothcomponents in the binding assay are cellular, binding can be assayedmicroscopically or by flow cytometry. See Kishimoto et al., supra. Whenone or both components is a purified protein, one component is usuallyimmobilized to a solid phase and the other labelled. Binding is thenassayed from label bound to the solid phase. Usually, the antibody ispreincubated with the source of P-selectin before adding the source ofcounterreceptor to the incubation mixture. Blocking activity is shownwhen an excess of antibody, i.e., 5-fold, 10-fold or up to 100-fold,substantially inhibits binding of P-selectin to its counterreceptor. Theprecise degree of inhibition will depend on the assay used. In an assaythat measures inhibition of platelet binding to HL-60 cells, an excessof P-selectin blocking antibodies typically exhibits at least 50, 60,70, 80 or 90% and usually about 80-90% inhibition.

The binding specificity of many blocking antibodies of the invention isfurther defined by their capacity to bind P-selectin in the complete orsubstantial absence of Ca⁺⁺ (e.g., in the presence of 2 mM EDTA (acalcium chelator) and the absence of Ca⁺⁺ in an in vitro assay). Bycontrast, most blocking antibodies against P-selectin isolated to daterequire Ca⁺⁺ for activity. See Geng et al., J. Biol. Chem.266:22313-22318 (1991). Antibodies requiring a Ca⁺⁺ cofactor forblocking activity may be less effective in in vivo conditions wherelevels of Ca⁺⁺ are expected to fluctuate.

The capacity of the antibodies of the invention to blockE-selectin-mediated functions can be demonstrated by analogous in vitroassays to those employed to show blocking of P-selectin mediatedfunctions. Suitable sources of E-selectin are mammalian cell linestransfected with E-selectin, activated endothelial cells, as well aspurified E-selectin (or extracellular domains thereof). If the assay isperformed using purified E-selectin, the E-selectin can be immobilizedto a solid support. Suitable sources of counterreceptors to E-selectinare leukocytes, neutrophils, monocytes, and HL-60 cells and appropriatecell lines transfected with L-selectin. The degree of binding inhibitionwill again depend on the components in the assay. In an assay thatmeasures binding between activated endothelial cells and HL-60 cells,the antibodies of the invention, when present in excess, typicallyexhibit at least about 20, 40, 60, 80% inhibition or more typicallyabout 25-75% or 50% inhibition.

The capacity of antibodies to block L-selectin mediated functions can bedemonstrated in a variety of in vitro assays. See, e.g., copendingapplication Ser. No. 08/160,516, filed Nov. 30, 1993 and Ser. No.08/160,074, filed Nov. 30, 1993 (incorporated by reference in theirentirety for all purposes). A simple visual assay for detecting suchinteraction has been described by Kishimoto et al., supra. Briefly,monolayers of human umbilical vein cells are stimulated with IL-1.Neutrophils, with or without pretreatment with the antibody under test,are added to the monolayer under defined conditions, and the number ofadhering neutrophils is determined microscopically. In one method, theneutrophils are obtained from human leukocyte adhesion deficientpatients. See Anderson et al., Ann. Rev. Med. 38:175 (1987). Theneutrophils from such patients lack integrin receptors, whose binding toneutrophils might obscure the effects of blocking L-selectin binding.

Preferred antibodies selectively bind a functional epitope on P-selectinand E-selectin molecules associated with a response to tissue injury andinflammation. Binding of the antibodies to a functional epitope onP-selectin and E-selectin effectively inhibits adhesion of leukocytes tothe activated vascular endothelium and/or to activated platelets invivo. Preferred antibodies impair the adhesion of leukocytes to theactivated vascular endothelium to prevent or inhibit an inflammatoryand/or thrombotic condition.

In vivo blocking efficacy can be demonstrated in the same animal modelsthat have been used to show efficacy for antibodies specific for asingle adhesion molecule. For example, Mulligan et al., 1991, 1992,supra, describe rat models to test the efficacy of antibodies inprotecting against lung injury; Coughlan et al., 1994, describe a ratmodel for testing the efficacy of antibodies in treatment of systemicendotoxemia; and Weyrich et al., supra, describe a cat model for testingthe protective effect of antibodies in myocardial ischemia andreperfusion injury. Other animal models for various inflammatorydiseases and disorders are described by Arfors et al., Blood 69:338(1987) (skin lesions); Tuomanen et al., J. Exp. Med. 170:959 (1989)(brain edema and death produced by bacterial meningitis); Lindbom etal., Clin. Immunol. Immunopath. 57:105 (1990) (tissue edema associatedwith delayed-type hypersensitivity reactions); Wegner et al., Science247:456 (1990) (airway hyperresponsiveness in allergic asthma); Goldmanet al., FASEB J. 5:A509 (1991) (remote lung injury followingaspiration); Gundel et al., J. Clin. Invest. 88:1407 (1991) (late-phasebronchoconstriction following antigen challenge); Hutchings et al.,Nature 346:639 (1990) (diabetes); Flavin et al., Transplant, Proc.23:533 (1991) (cardiac allograft survival); Wegner et al., Am. Rev.Respir. Dis. 143:A544 (1991) (lung damage and dysfunction secondary tooxygen toxicity); Cosimi et al., J. Immunol. 144:4604 (1990) (renalallograft rejection); Jasin et al., Arthritis Rheum. 33:S34 (1990)(antigen-induced arthritis); Thomas et al., FASEB J. 5:A509 (1991)(vascular injury and death in endotoxic shock); Bucky et al., Proc. Am.Burn Assoc. 23:133 (1991) (burns); Hernandez et al., Am. J. Physiol.253:H699 (1987) (permeability edema following ischemia reperfusion (IR)of intestine); Winquist et al., Circulation 82:III (1990); Ma et al.,Cir. Res. 82:III (1990) (myocardial damage following myocardialinfarction); Mileski et al., Surgery 108:206 (1990) (vascular and tissuedamage following hemorrhagic shock and resuscitation); Clark et al.,Stroke 22:877 (1991) (central nervous system damage following I/R of thespinal cord); Mileski et al., Proc. Am. Burn Assoc. 22:164 (1990) (edemaand tissue damage following frostbite and rewarming); Simpson et al.,Circulation 81:226 (1990) (infarct size following I/R of myocardium).Preferred antibodies show efficacy in at least one and usually severalof these inflammatory and thrombotic diseases and conditions.

Many of the blocking antibodies of the invention show the same orsimilar binding specificity as one of the exemplary antibodiesdesignated 5C7.29, 2C9.11 and 1D8.10. That is, the antibodies competewith at least one of the exemplified antibodies for specific binding toE-selectin and/or P-selectin. The E-selectin and P-selectin used in thetest is preferably human, and may be natural or recombinant. Competitionbetween antibodies is determined by an assay in which the immunoglobulinunder test inhibits specific binding of a reference antibody (e.g.,5C7.29) to an antigenic determinant on a P-selectin and/or E-selectinmolecule. Numerous types of competitive binding assays are known, forexample: solid phase direct or indirect radioimmunoassay (RIA), solidphase direct or indirect enzyme immunoassay (EIA), sandwich competitionassay (see Stahli et al., Methods in Enzymology 9:242-253 (1983)); solidphase direct biotin-avidin EIA (see Kirkland et al., J. Immunol.137:3614-3619 (1986)); solid phase direct labeled assay, solid phasedirect labeled sandwich assay (see Harlow and Lane, “Antibodies, ALaboratory Manual,” Cold Spring Harbor Press (1988)); solid phase directlabel RIA using I-125 label (see Morel et al., Molec. Immunol.25(1):7-15 (1988)); solid phase direct biotin-avidin EIA (Cheung et al.,Virology 176:546-552 (1990)); and direct labeled RIA (Moldenhauer etal., Scand. J. Immunol. 32:77-82 (1990)). Typically, such an assayinvolves the use of purified P-selectin or E-selectin bound to a solidsurface or cells bearing either of these, an unlabelled testimmunoglobulin and a labelled reference immunoglobulin. Competitiveinhibition is measured by determining the amount of label bound to thesolid surface or cells in the presence of the test immunoglobulin.Usually the test immunoglobulin is present in excess. Antibodiesidentified by competition assay (competing antibodies) includeantibodies binding to the same epitope as the reference antibody andantibodies binding to an adjacent epitope sufficiently proximal to theepitope bound by the reference antibody for steric hindrance to occur.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to P-selectin and/or E-selectinby at least 50 or 75%.

The antibodies of the invention usually exhibit a specific bindingaffinity for P-selectin and E-selectin of greater than or equal to about10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. However, antibodies do not necessarilyshow the same specific binding affinity for each of these ligands.Usually the upper limit of binding affinity of the antibodies is withina factor of about three, five or ten of that of one of the exemplifiedantibodies. Often the lower limit of binding affinity is also within afactor of about three, five or ten of that of the exemplifiedantibodies. The term “about” encompasses the degree of experimentalerror that may typically occur in the measurement of binding affinities.

A hybridoma producing the 5C7.29 antibody has been deposited with theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. under the Budapest Treaty on May 25, 1994 and given the AccessionNo. ATCC CRL 11640. The production of this antibody is described inExample 1.

C. Production of Antibodies

(1) Nonhuman Antibodies

Mouse, or other nonhuman antibodies crossreactive with P-selectin andE-selectin can be obtained using a variety of immunization strategies.In some strategies, nonhuman animals (usually nonhuman mammals), such asmice, are immunized with E-selectin and P-selectin antigens, eitherconcurrently or consecutively. In other strategies, nonhuman animals areimmunized with only one of these antigens. Preferred immunogens arecells stably transfected with P-selectin or E-selectin and expressingthese molecules on their cell surface. Other preferred immunogensinclude P-selectin and E-selectin proteins or epitopic fragments ofP-selectin and E-selectin containing the segments of these moleculesthat bind to the exemplified crossreacting antibodies.

Mouse or non-human antibodies crossreactive with all three selecting,i.e., P-selectin, E-selectin, and L-selectin, can be generated bysimilar strategies. Briefly, mice are immunized either simultaneously orsequentially with cells stably transfected with either P-selectin,E-selectin, or L-selectin, or purified selectin proteins or epitopicfragments thereof.

Antibody-producing cells obtained from the immunized animals areimmortalized and selected for the production of an antibody whichspecifically binds to multiple selectins. See generally, Harlow & Lane,Antibodies, A Laboratory Manual (C.S.H.P. N.Y., 1988) (incorporated byreference for all purposes). The binding assays for the differentselectins can be performed separately or concurrently. Concurrentanalysis is conveniently performed by two-color FACS screening afterincubation of hybridoma supernatants to cells transfected withselecting. For example, two populations of cells respectively expressingE-selectin and P-selectin are differentially labelled with a first labeland tested for capacity to bind hybridomas supernatants. Binding isdetected using an appropriate secondary antibody bearing a second label.This scheme is readily extendible to allow simultaneous detection ofbinding to all three selectins by differentially labelling threepopulations of cells respectively expressing E-selectin, P-selectin andL-selectin with different intensities of the first label. Alternatively,separate screening for E-selectin, P-selectin and, if desired,L-selectin binding, can be achieved by single color FACS analysis ofsupernatant binding to transfectant cells or by binding assay toimmobilized E-selectin, P-selectin, or L-selectin. Crossreactingantibodies are then further screened for their capacity to blockfunctional properties of E-selectin, P-selectin and L-selectin using thein vitro and in vivo assays described above. Most antibodies thatcrossreact with P-selectin or E-selectin also block the functionalcapacity of both of these molecules to interact with a counterreceptor.

(2) Humanized Antibodies

The invention provides humanized antibodies having similar bindingspecificity and affinity to selected mouse or other nonhuman antibodies.Humanized antibodies are formed by linking CDR regions (preferably CDR1,CDR2 and CDR3) of non-human antibodies to human framework and constantregions by recombinant DNA techniques. See Queen et al., Proc. Natl.Acad. Sci. USA 86:10029-10033 (1989) and WO 90/07861 (incorporated byreference in their entirety for all purposes). The humanizedimmunoglobulins have variable region framework residues substantiallyfrom a human immunoglobulin (termed an acceptor immunoglobulin) andcomplementarity determining regions substantially from a mouseimmunoglobulin described above, e.g., the 5C7.29 antibody (referred toas the donor immunoglobulin). The constant region(s), if present, arealso substantially from a human immunoglobulin.

In principal, a framework sequence from any human antibody may serve asthe template for CDR grafting. However, it has been demonstrated thatstraight CDR replacement onto such a framework often leads tosignificant loss of binding affinity to the antigen (Glaser et al., J.Immunol. 149: 2606 (1992); Tempest et al., Biotechnology 9: 266 (1992);Shalaby et al., J. Exp. Med. 17: 217 (1992)). The more homologous ahuman antibody is to the original murine antibody, the less likely willcombining the murine CDRs with the human framework be to introducedistortions into the CDRs that could reduce affinity. Therefore,homology (that is, percent sequence identity) of at least 65% betweenthe humanized antibody variable region framework and the donor antibodyvariable region framework is preferred.

The heavy and light chain variable region framework residues can bederived from the same or different human antibody sequences. However, aheavy chain and light chain framework sequences chosen from the samehuman antibody reduce the possibility of incompatibility in assembly ofthe two chains. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies. See Carter et al., WO 92/22653. Certain aminoacids from the human variable region framework residues can be selectedfor substitution based on their possible influence on CDR conformationand/or binding to antigen. Investigation of such possible influences isby modeling, examination of the characteristics of the amino acids atparticular locations, or empirical observation of the effects ofsubstitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine 5C7.29 variableregion framework residue and a selected human variable region frameworkresidue, the human framework amino acid should usually be substituted bythe equivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

(1) contacts antigen directly,

(2) is adjacent to a CDR region in the sequence, or

(3) otherwise interacts with a CDR region (e.g., is within about 4-6 Åof a CDR region).

Other candidates for substitution are acceptor human framework aminoacids that are unusual for a human immunoglobulin at that position.These amino acids can be substituted with amino acids from theequivalent position of the donor antibody or from the equivalentpositions of more typical human immunoglobulins. The variable regionframeworks of humanized immunoglobulins usually show at least 85%sequence identity to a human variable region framework sequence orconsensus of such sequences.

(3) Human Antibodies

In another aspect of the invention, human antibodies cross-reactive withE-selectin and P-selectin are provided. These antibodies are produced bya variety of techniques described below. Some human antibodies areselected by competitive binding experiments, or otherwise, to have thesame epitope specificity as an exemplified mouse antibody, such as5C7.29. Such antibodies are particularly likely to share similartherapeutic properties.

a. Trioma Methodology

The basic approach and an exemplary cell fusion partner, SPAZ-4, for usein this approach have been described by Oestberg et al., Hybridoma2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman etal., U.S. Pat. No. 4,634,666 (each of which is incorporated by referencein its entirety for all purposes). The antibody-producing cell linesobtained by this method are called triomas, because they are descendedfrom three cells—two human and one mouse. Initially, a mouse myelomaline is fused with a human B-lymphocyte to obtain anon-antibody-producing xenogeneic hybrid cell, such as the SPAZ-4 cellline described by Oestberg, supra. The xenogeneic cell is then fusedwith an immunized human B-lymphocyte to obtain an antibody-producingtrioma cell line. Triomas have been found to produce antibody morestably than ordinary hybridomas made from human cells.

The B-lymphocytes are obtained from the blood, spleen, lymph nodes orbone marrow of a human donor. In vivo immunization of a living humanwith E-selectin and/or P-selectin is usually undesirable because of therisk of initiating a harmful response. Thus, B-lymphocytes are usuallyimmunized in vitro with an E-selectin and/or P-selectin or an antigenicfragment of either of these, or a cell bearing either of these. Specificepitopic fragments consisting essentially of the amino acid segmentsthat bind to one of the exemplified murine antibodies are preferred forin vitro immunization. B-lymphocytes are typically exposed to antigenfor a period of 7-14 days in a media such as RPMI-1640 (see Engleman,supra) supplemented with 10% human serum.

The immunized B-lymphocytes are fused to a xenogeneic hybrid cell suchas SPAZ-4 by well known methods. For example, the cells are treated with40-50% polyethylene glycol of MW 1000-4000, at about 37 degrees, forabout 5-10 min. Cells are separated from the fusion mixture andpropagated in media selective for the desired hybrids (e.g., HAT or AH).Clones secreting antibodies having the required binding specificity areidentified by assaying the trioma culture medium for the ability to bindto E-selectin and P-selectin using the same methods as discussed abovefor nonhuman antibodies. Triomas producing human antibodies having thedesired specificity are subcloned by, e.g., the limiting dilutiontechnique and grown in vitro in culture medium.

Although triomas are genetically stable they may not produce antibodiesat very high levels. Expression levels can be increased by cloningantibody genes from the trioma into one or more expression vectors, andtransforming the vector into a cell line such as the cell linesdiscussed, infra, for expression of recombinant or humanizedimmunoglobulins.

b. Transgenic Non-Human Mammals

Human antibodies crossreactive with P-selectin and E-selectin can alsobe produced from non-human transgenic mammals having transgenes encodingat least a segment of the human immunoglobulin locus. Usually, theendogenous immunoglobulin locus of such transgenic mammals isfunctionally inactivated. Preferably, the segment of the humanimmunoglobulin locus includes unrearranged sequences of heavy and lightchain components. Both inactivation of endogenous immunoglobulin genesand introduction of exogenous immunoglobulin genes can be achieved bytargeted homologous recombination, or by introduction of YACchromosomes. The transgenic mammals resulting from this process arecapable of functionally rearranging the immunoglobulin componentsequences, and expressing a repertoire of antibodies of various isotypesencoded by human immunoglobulin genes, without expressing endogenousimmunoglobulin genes. The production and properties of mammals havingthese properties are described in detail by, e.g., Lonberg et al.,WO93/12227 (1993); Kucherlapati, WO 91/10741 (1991) (each of which isincorporated by reference in its entirety for all purposes). Transgenicmice are particularly suitable. Crossreacting P-selectin/E-selectinhuman antibodies are obtained by immunizing a transgenic nonhumanmammal, such as described by Lonberg or Kucherlapati, supra, accordingto the same strategy as discussed for a nontransgenic nonhuman animal(section I.C.(1)). Monoclonal antibodies are prepared by, e.g., fusingB-cells from such mammals to suitable myeloma cell lines usingconventional Kohler-Milstein technology.

c. Phage Display Methods

A further approach for obtaining human crossreacting antibodies toE-selectin and P-selectin is to screen a DNA library from human B cellsas described by Dower et al., WO 91/17271 and McCafferty et al., WO92/01047 (each of which is incorporated by reference in its entirety forall purposes). In these methods, libraries of phage are produced inwhich members display different antibodies on their outer surfaces.Antibodies are usually displayed as Fv or Fab fragments. Phagedisplaying antibodies are selected by affinity enrichment for binding toeither P-selectin or E-selectin. Phage identified by the initial screenare then further screened for crossreaction with the other ligand.

In a variation of the phage-display method, human antibodies having thebinding specificity of a selected murine antibody can be produced. SeeWinter, WO 92/20791. In this method, either the heavy or light chainvariable region of the selected murine antibody (e.g., 5C7.29) is usedas a starting material. If, for example, a light chain variable regionis selected as the starting material, a phage library is constructed inwhich members displays the same light chain variable region (i.e., themurine starting material) and a different heavy chain variable region.The heavy chain variable regions are obtained from a library ofrearranged human heavy chain variable regions. A phage showing strongspecific binding for P-selectin and E-selectin (e.g., at least 10⁸ andpreferably at least 10⁹ M⁻¹) is selected. The human heavy chain variableregion from this phage then serves as a starting material forconstructing a further phage library. In this library, each phagedisplays the same heavy chain variable region (i.e., the regionidentified from the first display library) and a different light chainvariable region. The light chain variable regions are obtained from alibrary of rearranged human variable light chain regions. Again, phageshowing strong specific binding for P-selectin and E-selectin areselected. These phage display the variable regions of completely humanantibodies that crossreact with E-selectin and P-selectin. Theseantibodies usually have the same or similar epitope specificity as themurine starting material (e.g., 5C7.29).

D. Bispecific Antibodies

The invention also provides bispecific or bifunctional antibodies thathave one binding site that specifically binds to P-selectin andE-selectin and a second binding site that specifically binds to a secondmoiety. In bispecific antibodies, one heavy and light chain pair isusually from a crossreacting antibody and the other pair from anantibody raised against another epitope. This results in the property ofmulti-functional valency, i.e., ability to bind at least two differentepitopes simultaneously, one of which is the epitope to which the antiP-selectin/E-selectin crossreacting antibody binds. The other epitopecould be e.g., an epitope on L-selectin.

E. Other Therapeutic Agents

Having produced an antibody having desirable properties, such as 5C7.29and the other exemplified antibodies, other non antibody agents havingsimilar binding specificity/and or affinity can be produced by a varietyof methods. For example, Fodor et al., U.S. Pat. No. 5,143,854, discussa technique termed VLSIPS™, in which a diverse collection of shortpeptides are formed at selected positions on a solid substrate. Suchpeptides could then be screened for binding to an epitopic fragmentrecognized by 5C7.29, optionally in competition with the 5C7.29.Libraries of short peptides can also be produced using phage-displaytechnology, see, e.g., Devlin WO91/18980. The libraries can be screenedfor binding to an epitopic fragment recognized by e.g., 5C7.29,optionally in competition with 5C7.29.

II. Nucleic Acids

The genes encoding the heavy and light chains of immunoglobulinsproduced by hybridoma or trioma cell lines secreting crossreactingantibodies are cloned according to methods described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor,N.Y., 1989); Berger & Kimmel, Methods in Enzymology, Volume 152, Guideto Molecular Cloning Techniques (Academic Press, Inc., San Diego,Calif., 1987); Co et al., J. Immunol. 148:1149 (1992). For example,genes encoding heavy and light chains are cloned from a hybridomalsgenomic DNA or cDNA produced by reverse transcription of RNA. Cloning isaccomplished by conventional techniques including the use of PCR primersthat hybridize to the sequences flanking or overlapping the genes, orsegments of genes, to be cloned.

Typically, recombinant constructs comprise DNA segments encoding acomplete human immunoglobulin heavy chain and/or a complete humanimmunoglobulin light chain of an immunoglobulin expressed by a hybridomaor trioma cell line. Alternatively, DNA segments encoding only a portionof the primary antibody genes are produced, which portions possessbinding and/or effector activities. Other recombinant constructs containsegments of immunoglobulin genes fused to segments of otherimmunoglobulin genes, particularly segments of other human constantregion sequences (heavy and/or light chain). Human constant regionsequences can be selected from various reference sources, includingthose listed in Kabat et al., supra.

DNA segments encoding crossreacting P-selectin/E-selectin antibodies canbe modified by recombinant DNA techniques such as site-directedmutagenesis (see Gillman & Smith, Gene 8:81-97 (1979); Roberts et al.,Nature, 328:731-734 (1987). Such modified segments will usually retainantigen binding capacity and/or effector function. Moreover, themodified segments are usually not so far changed from the originalsequences to prevent hybridization to these sequences under stringentconditions. The modified segments will usually encode an immunoglobulinshowing substantial sequence identity to a reference immunoglobulin fromwhich it was derived. Because, like many genes, immunoglobulin genescontain separate functional regions, each having one or more distinctbiological activities, the genes may be fused to functional regions fromother genes to produce fusion proteins (e.g., immunotoxins) having novelproperties or novel combinations of properties.

The recombinant polynucleotide constructs will typically include anexpression control sequence operably linked to the coding sequences,including naturally-associated or heterologous promoter regions.Preferably, the expression control sequences will be eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells. Once the vector has been incorporated into the appropriatehost, the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and the collection andpurification of the crossreacting antibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors will contain selection markers, e.g.,ampicillin-resistance or hygromycin-resistance, to permit detection ofthose cells transformed with the desired DNA sequences.

E. coli is one prokaryotic host particularly useful for cloning the DNAsequences of the present invention. Microbes, such as yeast are alsouseful for expression. Saccharomzyces is a preferred yeast host, withsuitable vectors having expression control sequences, an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

Mammalian cells are a preferred host for expressing nucleotide segmentsencoding immunoglobulins or fragments thereof. See Winnacker, From Genesto Clones, (VCH Publishers, N.Y., 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped in the art, and include CHO cell lines, various COS celllines, HeLa cells, L cells and myeloma cell lines. Preferably, the cellsare nonhuman. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromendogenous genes, cytomegalovirus, SV40, adenovirus, bovinepapillomavirus, and the like. See Co et al., J. Immunol. 148:1149(1992).

The vectors containing the DNA segments of interest can be transferredinto the host cell by well-known methods, depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment,electroporation, lipofection, biolistics or viral-based transfection maybe used for other cellular hosts. Other methods used to transformmammalian cells include the use of polybrene, protoplast fusion,liposomes, electroporation, and microinjection (see generally, Sambrooket al., supra).

Once expressed, crossreacting immunoglobulins of the invention can bepurified according to standard procedures of the art, including HPLCpurification, column chromatography, gel electrophoresis and the like(see generally, Scopes, Protein Purification (Springer-Verlag, N.Y.,1982)).

III. Epitope Mapping

The P-selectin epitope(s) bound by the 5C7.29 or other crossreactingantibody can be determined by providing a family of fragments containingdifferent amino acid segments from P-selectin. Each fragment typicallycomprises at least 4, 6, 8, 10, 20, 50 or 100 contiguous amino acids.The family of polypeptide fragments cover much or all of the amino acidsequence of the extracellular domain of a P-selectin polypeptide.Members of the family are tested individually for binding to e.g., the5C7.29 antibody. The smallest fragment that can specifically bind to theantibody being tested contains the amino acid sequence of the epitoperecognized by the antibody. The E-selectin epitope bound by the antibodyis mapped by an analogous strategy using a family of peptides fromE-selectin. The respective epitopes on P-selectin and E-selectin areexpected to map to segments of these molecules showing a high degree ofsequence identity. The epitopic fragments are useful as immunogens forgenerating further crossreacting antibodies. The epitopic fragments arealso useful as therapeutic agents that agonize or antagonize thefunction of P-selectin or E-selectin.

Another method to map epitopes involves testing the ability of anantibody to bind to E-selectin or P-selectin to which random mutationshave been introduced. This method is described in more detail in Example9.

IV. Pharmaceutical Compositions

The pharmaceutical compositions for use in the therapeutic methodsdiscussed infra, typically comprise an active agent, such ascrossreacting E-selectin/P-selectin antibody, dissolved in an acceptablecarrier, preferably an aqueous carrier. Some compositions contain acocktail of multiple active agents, for example, a crossreactingantibody and a thrombolytic agent. A variety of aqueous carriers can beused, e.g., water, buffered water, phosphate buffered saline (PBS), 0.4%saline, 0.3% glycine, human albumin solution and the like. Thesesolutions are sterile and generally free of particulate matter. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride and sodium lactate. The concentration of antibody inthese formulations can vary widely, i.e., from less than about 0.005%,usually at least about 1% to as much as 15 or 20% by weight and will beselected primarily based on fluid volumes, viscosities, and so forth, inaccordance with the particular mode of administration selected.

Thus, a typical pharmaceutical composition for injection could be madeup to contain 1 ml sterile buffered water, and 1-10 mg ofimmunoglobulin. A typical composition for intravenous infusion could bemade up to contain 250 ml of sterile Ringer's solution, and 150 mg ofantibody. Methods for preparing parenterally administrable compositionsare described in Remington's Pharmaceutical Science (15th ed., MackPublishing Company, Easton, Pa., 1980), which is incorporated byreference in its entirety for all purposes.

Therapeutic agents of the invention can be frozen or lyophilized forstorage and reconstituted in a suitable carrier prior to use.Lyophilization and reconstitution can lead to varying degrees ofantibody activity loss (e.g., with conventional immune globulins, IgMantibodies tend to have greater activity loss than IgG antibodies).Dosages may have to be adjusted to compensate.

V. Therapeutic Methods

The antibodies of the present invention are useful for treatment ofinflammatory diseases and conditions, especially those which aremediated by neutrophils. The dual specificity of the antibodies leads tothe inhibition of inflammatory events mediated by either P-selectin orE-selectin.

For example, the antibodies are suitable for therapeutic andprophylactic treatment of ischemia-reperfusion injury caused bymyocardial infarction, cerebral ischemic event (e.g., stroke), renal,hepatic or splenial infarction, brain surgery, lung injury, shock,cardiac surgery (e.g., coronary artery bypass), elective angioplasty,and the like. Other preferred applications are the treatment of sepsis,adult respiratory distress syndrome, and multiple organ failure. Theantibodies are also useful for treating injury due to trauma, burns,frostbite or damage to the spinal cord. The antibodies will also finduse in treating autoimmune diseases including rheumatoid arthritis,systemic lupus erythematosus, multiple sclerosis, type I diabetes anduveitis, in treating inflammatory diseases of the skin such aspsoriasis, and in treating meningitis and encephalitis. The antibodiesare also useful for treating allergic rhinitis, asthma and anaphylaxis.Other typical applications are the prevention and treatment of organtransplant rejection and graft-versus-host disease.

The pharmaceutical compositions containing the antibodies areparticularly useful for parenteral administration, i.e., subcutaneously,intramuscularly or intravenously. The antibodies of the invention mayalso be administered, typically for local application, by gavage orlavage, intraperitoneal injection, ophthalmic ointment, topicalointment, intracranial injection (typically into a brain ventricle),intrapericardiac injection, or intrabursal injection.

The compositions containing the present antibodies or a cocktail thereofcan be administered for prophylactic and/or therapeutic treatments. Intherapeutic applications, compositions are administered to a patientalready suffering from an inflammatory disease, in an amount sufficientto cure or at least partially arrest the disease and its complications.An amount adequate to accomplish this is defined as a “therapeuticallyeffective dose.” Amounts effective for this use will depend upon theseverity of the disease and the general state of the patient's ownimmune system, but generally range from about 1 to about 200 mg ofantibody per dose, with dosages of from 5 to 80 mg per patient beingmore commonly used. Dosing schedules will vary with the disease stateand status of the patient, and will typically range from a single bolusdosage or continuous infusion to multiple administrations per day (e.g.,every 4-6 hours), or as indicated by the treating physician and thepatient's condition. In life-threatening or potentially life-threateningsituations, it is possible and may be felt desirable by the treatingphysician to administer substantial excesses of these antibodies.

In prophylactic applications, compositions containing the presentantibodies or a cocktail thereof are administered to a patient notalready suffering from a particular disease to enhance the patient'sresistance. Such an amount is defined to be a “prophylacticallyeffective dose.” In this use, the precise amounts again depend upon thepatient's state of health and general level of immunity, but generallyrange from 1 to 80 mg per dose. Preferred prophylactic uses are for theprevention of adult respiratory distress syndrome in patients alreadysuffering from sepsis or trauma; prevention of organ transplantrejection; and prevention of reperfusion injury in patients sufferingfrom ischemia. In seriously ill patients, dosages of about 50 to 150 mgof humanized or human immunoglobulin per administration are frequentlyused, and larger dosages may be indicated.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of the antibody(ies) of this invention sufficient to treatthe patient effectively.

The antibodies can also be used in combination with other antibodies,particularly antibodies reactive with different adhesion molecules. Forexample, suitable antibodies include those specific for CD11a, CD11b,CD18, L-selectin, and ICAM-1. Other suitable antibodies are thosespecific for lymphokines, such as IL-1, IL-2 and IFN-γ, and theirreceptors. The antibodies of the invention can also be administered inconjunction with chemotherapeutic agents. Suitable agents includenon-steroidal anti-inflammatory drugs and corticosteroids, but numerousadditional agents (e.g., cyclosporin) can also be used.

In some therapeutic methods of ischemia-reperfusion therapy,crossreacting antibodies are used in combination with thrombolyticagents. In previous methods, patients with myocardial infarction orunstable angina are often treated by opening the occluded coronaryartery. Reopening of the obstructed coronary artery can be achieved byadministration of thrombolytic agents which lyse the clot causing theobstruction, and which, thereby, restore coronary blood flow.Reperfusion of the vessel can also be achieved by percutaneoustransluminal coronary angioplasty (PTCA) by means of balloon dilation ofthe obstructed and narrowed segment of the coronary artery. However,restoration of coronary blood flow leads to ischemia-reperfusion injuryin prior methods.

In the present methods, ischemia-reperfusion injury is reduced orprevented by combination of a thrombolytic agent or of PTCA withcrossreacting E-selectin/P-selectin antibodies. Antibodies are usuallyadministered prophylactically before, or at the same time as,administration of thrombolytic agents or initiation of PTCA. Furtherdoses of antibody are then often administered during and afterthrombolytic or angioplastic treatment. The interval betweenprophylactic administration of the antibodies and initiation ofthrombolytic or angioplastic treatment is usually 5-60 mins, preferably5-30 min, and most preferably 5-10 min. The antibodies are administeredparentally, preferably by intravenous injection, in doses of 0.01-10mg/kg body weight, preferably of 0.14-5 mg/kg and most preferably of0.3-3 mg/kg. The antibodies can be given as an intravenous bolusinjection, e.g., over 1-5 min., as repeated injections of smaller doses,or as an intravenous infusion. The bolus injection is especially usefulfor the prophylactic dose or in an emergency. Further doses ofantibodies can be repeated (e.g., every 4-24 hr) during and afterthrombolytic or angioplastic treatment of acute myocardial infarction atthe same proportions as described above to achieve optimal plasma levelsof the antibody.

Thrombolytic agents are drugs having the capacity, directly orindirectly, to stimulate dissolution of thrombi in vivo. Thrombolyticagents include tissue plasminogen activator (see EP-B 0 093 619),activase, alteplase, duteplase, silteplase, streptokinase, anistreplase,urokinase, heparin, warfarin and coumarin. Additional thrombolyticagents include saruplase and vampire bat plasminogen activator. SeeHarris, Protein Engineering 6:449-458 (1987); PCT/EP 90/00194; U.S. Pat.No. 4,970,159. Thrombolytic agents are administered to a patient in anamount sufficient to partially disperse, or prevent the formation of,thrombi and their complications. An amount adequate to accomplish thisis defined as a “therapeutically effective dose” or “efficacious dose.”Amounts effective for this use will depend upon the severity of thecondition, the general state of the patient, the route of administrationand combination with other drugs. Often, therapeutically effective dosesof thrombolytic agents and administration regimens for such agents withcrossreacting antibodies to E-selectin and P-selectin are those approvedby the FDA for independent uses of thrombolytic agents, e.g., 100 mg ofalteplase or 1.5 million IU of streptokinase.

VI. Methods of Diagnosis

The monoclonal antibodies of the present invention are useful fordiagnosing the inflammatory conditions discussed above and monitoringthe treatment thereof. The antibodies detect P-selectin and E-selectinin a tissue sample such as serum or endothelial cells, e.g., by ELISA orRIA. The presence of either selectin is diagnostic of inflammation.Selectin levels may be employed as a differentiation marker to identifyand type cells of certain lineages and developmental origins.

In such procedures, the antibody can be labelled directly (e.g., byradioactive or fluorescent label) and immune complexes detected via thelabel. Usually, however, the antibody is unlabelled and the desiredantigen-monoclonal antibody complex is detected with anenzyme-conjugated antibody against the monoclonal antibody. Diagnosiscan also be achieved by in vivo administration of a labelledcrossreacting P-selectin/E-selectin antibody and detection by in vivoimaging. The concentration of antibody administered should be sufficientthat the binding to cells having the target antigen is detectablecompared to the background signal. The diagnostic reagent can belabelled with a radioisotope for camera imaging, or a paramagneticisotope for magnetic resonance or electron spin resonance imaging.

VII. Other Uses

The antibodies are also useful for affinity purification of selectinsand cells expressing the same on their external surfaces. The antibodiescan also be used. to generate anti-idiotypic antibodies that mimic aselectin domain responsible for antibody binding. Anti-idiotypicantibodies are useful as competitive inhibitors of selectin binding. Forexample, an anti-idiotypic antibody to a crossreacting P-selectin,E-selectin monoclonal antibody can be selected to compete withP-selectin and/or E-selectin for binding to their counterreceptors. Theantibodies are also useful in screening for a therapeutic agent havingthe same binding specificity as a crossreacting antibody (see Section I.E).

The following examples are provided to illustrate but not to limit theinvention:

EXAMPLE 1 Preparation of Cells Transfected With Selectins

L1-2 murine pre-B cell selectin transfectants are obtained by insertingthe respective human selectin genes downstream of the LCMV promoter inpMRB101 or similar plasmid (pMRB101 is a derivative of EEb whichcontains the E. coli gpt gene. Mulligan et al., Proc. Natl. Acad. Sci.USA 78:2072-2076 (1981); Stephans et al., Nucleic Acids Research 17:7110(1989)). Plasmid DNA is introduced into L1-2 cells by standard methods,such as electroporation, and the cells are selected for resistance tomycophenolic acid. Cells expressing high levels of the appropriateselectin are further selected by “panning” or fluorescence activatedcell sorting techniques. See Lymphocytes, A Practical Approach (G.C.B.Klaus, IRL Press, Oxford, England, 1987).

EXAMPLE 2 Production of Crossreacting Monoclonal Antibodies

Crossreacting antibodies were produced using two different immunizationprocedures. In all of these procedures, the inoculum was 10⁷ L1-2selectin transfectant cells (Berg et al., 1991, 1992, supra) in PBS perinjection into mice. In one procedure, Balb/c mice at 4-6 weeks of age(Simonson Labs, Gilroy, Calif.) were injected IP with L1-2^(E-selectin)transfectants at day 0 and day 14, and L1-2^(P-selectin) transfectantsat day 46, followed by fusion of spleen cells on day 50. In a secondprocedure, C57/Ld mice at 4-6 weeks of age (Jackson Labs, Bar Harbor,Me.) were immunized in the footpad with hypotonically lysedL1-2^(E-selectin) cells on day 0, then with intact L1-2^(E-selectin)cells on days 3 and 6, and with L1-2^(P-selectin) cells on day 9. Thedraining lymph node lymphocytes were fused on day 12. In each procedure,mouse B-cells were fused with P3X mouse myeloma cells using polyethyleneglycol.

Hybridoma supernatants were screened for specific binding to both E- andP-selectin by two-color FACS analysis. L1-2^(P-selectin) andL1-2^(control) transfectants were biotinylated by incubation with aminohexanoyl-biotin-N-hydroxy succinimide (Zymed Labs, South San Francisco,Calif.) at 10 μg/ml in PBS pH 8.0 for 25 min, at RT. After washing,2×10⁷ cells/ml were incubated with FITC-Z-Avidin (Zymed Labs, So. SanFrancisco, Calif.) diluted 1:150 for L1-2^(P-selectin) cells and 1:1000for L1-2^(control) cells in FACS Buffer (2% BSA/PBS/10 mM NaN₃) for 30min at 4° C. After washing, cells were mixed with unlabelledL1-2^(E-selectin) cells at a 1:1:1 ratio in FACS Buffer. 50 μl hybridomasupernatants were added to 200,000 mixed cells in 50 μl in 96-wellplates and incubated for 1 hr on ice. After washing, secondary agent wasadded, 50 μl of 1:500 Goat F(ab′)2 anti-mouse IgG-PE conjugated (TAGO,Burlingame, Calif.) for 30 min prior to washing and fixation. FACSanalysis was performed on a Becton Dickinson FACScan™ (San Jose,Calif.), according to standard procedures.

Supernatants containing antibodies reacting with both P-selectin andE-selectin were identified by a shift in red fluorescence of theL1-2^(E-selectin) transfectant (unlabelled with FITC) and the brightestFITC labelled cells (L1-2^(P-selectin) transfectants). The control L1-2cells (moderately labelled with FITC) did not show a shift in redfluorescence, indicating that binding was specific for P-selectin andE-selectin. The yield of crossreacting antibodies as a ratio ofsupernatants screened was {fraction (1/844 )}and {fraction (2/57 )}forthe two immunization schedules.

Supernatants showing binding to P-selectin and E-selectin transfectantswere subcloned by limiting dilution and grown in serum free mediumcontaining residual amounts of FBS. Three E-/P-selectin cross-reactingantibodies, designated 5C7.29, 1D8.10 and 2C9.11, were purified fromthese supernatants on Protein A-Sepharose™ (Pierce) according to therecommended protocol. Two antibodies reacting only with E-selectin, 1E4and 2D4, and an antibody reacting only with P-selectin, 5F4, wereidentified by the same method. The isotypes of 5C7.29, 1D8.10, 2C9.11,1E4, and 5F4 were determined to be IgG1, and that of 2D4 was determinedto be IgG2a using an Innogenetics Inno-Lia mouse monoclonal antibodyisotyping kit (Biosource International, Camarillo, Calif.).

The three E-/P-selectin crossreacting-antibodies were also tested fortheir ability to bind to the natural ligands, rather than therecombinant forms used in the initial screening assays, by single colorFACS analysis. The source of natural E-selectin used in these tests wasTNF-α-activated human umbilical vein endothelial cells (HUVEC). Inactivated form, HUVEC cells express E-selectin, but do not expressappreciable amounts of P-selectin. FIG. 1b shows that theE-/P-cross-reactive antibody 5C7.29 reacts with TNF-α activated HUVEC(shown by black histograms) but not unactivated HUVEC (grey histograms).Similar results were obtained for the two other cross-reactingantibodies 2C9.11 and 1D8.10. The activated cells also reacted with theanti-E-selectin blocking antibody H18/7 (FIG. 1a) (Becton Dickinson (SanJose, Calif.)), but not with P-selectin-specific antibodies WAPS 12.2and 5F4. (WAPS 12.2, a P-selectin blocking antibody, was provided by R.Aaron Warnock and Eugene C. Butcher (Stanford, Calif.).)

The source of natural P-selectin used in these tests wasthrombin-activated platelets. FIG. 2b shows that 5C7.29 binds to thesecells as does the known P-selectin antibody WAPS 12.2 (FIG. 2a). Similarresults were obtained with 2C9.11 and 1D8.10. Platelets did notsignificantly react with anti-E-selectin antibodies H18/7 or 1E4.

The E-/P-selectin crossreacting antibodies were further analyzed forbinding to L1-2^(L-selectin) transfectants, and with normal humanlymphocytes. Specific binding was not observed, demonstrating that theantibodies are specific for E- and P-selectins and do not bind toL-selectin.

To confirm that the crossreacting antibodies were truly monoclonal,preclearing experiments were performed. 10 ng antibody (a limitingamount) was incubated with a large number (10⁷) of L1-2^(E-selectin)cells or L1-2^(P-selectin) cells for 1 hr. The supernatant was thentransferred to a second aliquot of L1-2^(E-selectin) cells orL1-2^(P-selectin) cells (the same cell type as before) and incubated for1 hr. Supernatant was transferred to a third aliquot of cells of thesame type as before for a further 1 hr incubation. Supernatant was thenremoved and examined for reactivity with L1-2^(E-selectin),L1-2^(P-selectin) or L1-2 untransfected cells by one-color FACSanalysis.

FIG. 3 shows that preincubation of a solution of the 5C7.29 antibodywith L1-2^(P-selectin) transfectants eliminated subsequent reactivityfor both P-selectin and E-selectin. Similar results were found followingpreincubation with L1-2^(E-selectin) transfectants. These results wouldbe obtained only if the antibody bound to both selectins, and not if theantibody were a mixture of two different antibodies, one reactive withE-selectin and one reactive with P-selectin. Therefore, the dualspecificities of 5C7.29 reside in the same antibody. Similar resultswere obtained for the 2C9.11 and 1D8.10 antibodies.

EXAMPLE 3 Inhibition of E-Selectin-Mediated Functions

The antibody 5C7.29 was tested for the ability to block E-selectinmediated functions. In one assay, the antibody was tested for inhibitionof HL-60 binding to tumor necrosis factor-α (TNF-α) activated humanumbilical vein endothelial cells (HUVEC). This binding assay simulatesthe binding of neutrophils to endothelial cells in an inflammatoryresponse. The HL-60 cells are a promyelocytic cell line derived from apatient with acute promyelocytic leukemia. Collins et al., Nature 270,347-349 (1977). The HUVEC cells are endothelial cells that whenactivated with TNF-α for 4-6 hours express E-selectin, and notP-selectin.

HUVEC were obtained from Clonetics (San Diego, Calif.) and cultured assuggested. Confluent cultures, up to passage 6, grown in 8 well plasticLab Tek slides (Nunc, Naperville, Ill.) were activated for 4 hours with1 ng/ml TNF-α (R&D Systems, Minneapolis, Minn.). HUVEC cultures werewashed and incubated in 0.15 ml Assay Buffer (10% normal bovineserum/10% normal rabbit serum/10 mM HEPES, pH 7.2/RPMI) containingantibodies at 17 μg/ml (i.e., in excess) for 20 min.

HL-60 cells were fluorescently labelled with 6-carboxyfluoresceindiacetate acetoxy-methyl ester (CFDA-AM, Molecular Probes, Eugene OR)(von Andrian et al., 1991, supra) by a 30 min incubation in 10 mg/mlRPMI/10 mM HEPES, pH 7.2, washed and resuspended in Assay Buffer andincubated at RT for 20 min. The resuspended cells (6×10⁵ cells in 0.15ml) were then added to the HUVEC cultures.

Slides were rotated at 50 rpm on a rotator (Innova 200, New BrunswickInc.) for 15 min at RT. The cover slips were removed and non-adherentHL-60 cells washed off by dipping slides in DMEM. Adherent cells werefixed by immersion in 1% paraformaldehyde-PBS. Slides were examinedmicroscopically and the number of bound cells per field determined. Twotreatments per slide (in quadruplicate) were analyzed.

FIG. 4 shows that the number of HL-60 cells binding to the activatedHUVEC was decreased 47% by preincubation with 5C7.29. This comparedfavorably with blocking by the anti-E-selectin-specific antibody H18/7(38%). Binding was not significantly reduced by a control antibody.

Because HUVEC can also express P-selectin (although only at low levelsunder the present activation conditions), 5C7.29 was also tested forHL-60 binding to CHO cells transfected with E-selectin. CHO cellspermanently transfected with a truncated form of E-selectin containingthe first four N-terminal domains of E-selectin fused to thetransmembrane and cytoplasmic domain of another protein were producedaccording to standard methods. Expression was confirmed by reactivitywith a control anti-E-selectin antibody (H18/7). Inhibition of bindingbetween fluorescently labelled HL-60 and the transfected CHO cells wasperformed using the same assay as for the TNF-α-activated HUVEC. 5C7.29was found to block adhesion by 82% (FIG. 5). Similar results wereobserved with 1D8.10, 2C9.11 and the E-selectin blocking antibody 1E4.The non-blocking P-selectin specific control antibody 5F4 had nosignificant effect in this assay.

The cross-reacting antibodies also blocked normal human peripheral bloodneutrophil binding to TNF-α-activated HUVEC. At a final concentration of10 μg/ml, 5C7.29 blocked 71 +/−13%, 2C9.11 blocked 62 +/−8% and 1D8blocked 52 +/−10% of neutrophil binding to activated HUVEC, while theanti-E-selectin antibodies 1E4 and H18/7 (Bevilacqua et al., 1987,supra) blocked 68 +/−4% and 68 +/−15%, and a control mouse IgG1 antibodydid not block (−21% +/−11%), n=4. For these experiments, neutrophilswere isolated from normal human blood by density gradient centrifugationand dextran sedimentation by standard procedures (Current Protocols inImmunology, Coligan et al., eds., John Wiley and Sons, New York, 1992).Assays were performed as for HL-60 cells except neutrophils were addedto HUVEC at 7.5×10⁴ in 0.15 ml.

EXAMPLE 4 Inhibition of P-selectin-Mediated Functions

The antibodies 5C7.29, 2C7.11 and 1D8.10 were tested for their abilityto block P-selectin-mediated functions. Blocking was tested in aplatelet-HL-60 rosette assay (Corral et al., 1990, supra). The plateletsprovide a source of cells expressing P-selectin and the HL-60 cellssimulate neutrophils. Normal human blood was collected with sodiumcitrate as anticoagulant and the platelet-rich plasma (PRP) prepared bycentrifugation at 250g for 10 min. Platelets were isolated from PRP bycentrifugation at 100 g for 20 min and resuspended at 3×10⁸/ml in PBS,pH 7.2. Monoclonal antibodies (1 μg in 20 μl, i.e., an excess) wereadded to 20 μl platelets. In some experiments normal human thrombin (0.3U/μl) was added to activate the platelets as described by Corral et al.,1990, supra. After 45 min, 20 μl HL-60 cells (10⁶/ml in PBS) were addedand further incubated for 45 min. Bound platelets were fixed to HL-60cells by addition of glutaraldehyde to 1.25%. At least 100 HL-60 cellsfor each sample were observed microscopically and the number of cellswith bound platelets (>2 platelets per HL-60 cell) determined.

FIG. 6 shows that all three crossreacting antibodies block rosetting toabout the same extent as the P-selectin specific blocking antibody WAPS12.2. Similar blocking experiments can be performed using humanperipheral blood neutrophils in place of HL-60 cells. Neutrophils areprepared by the same method and used at the same concentration asdescribed in Example 3.

EXAMPLE 5 Cloning and Sequencing of Mouse 5C7.29 Heavy Chain and LightChain Variable Region cDNA

cDNAs for the heavy chain and light chain variable region genes of themouse 5C7.29 antibody were cloned using anchored polymerase chainreactions as described (see Co et al., J. Immunol. 148: 1149 (1992)),using 3′ primers that hybridized to the constant regions and containedHindIII sites, and 5′ primers that hybridized to the dG tails andcontained EcoRI sites. The PCR amplified fragments were digested withEcoRI and HindIII and cloned into the pUC18 or pUC19 vectors forsequencing. At least two gamma-1 specific and two kappa specific cloneswere sequenced. The gamma-1 clones and the kappa clones are respectivelyidentical in sequence. The variable region cDNA sequences and thededuced amino acid sequences for the gamma-1 and kappa chains are shownin FIGS. 7A-7B [SEQ ID NOS:1-4].

EXAMPLE 6 Design of Humanized 5C7.29 Antibody Variable Domain

Based on a sequence homology search against the NBRF protein sequencedatabase, the variable regions of light chain subclass I and heavy chainsubclass III show good homology to the mouse 5C7.29 antibody. Inparticular, the antibody III-3R provides the best framework homologywith 5C7.29 and was chosen to provide the framework sequences forhumanization of 5C7.29. However, other members of the light chainsubclass I and heavy chain subclass III would also be especiallysuitable for use in providing the frameworks of the respective humanized5C7.29 chains.

The computer program ENCAD (M. Levitt et al., J. Mol. Biol. 168: 595(1983)) was used to construct a molecular model of the 5C7.29 variabledomain. The program ABMOD (B. T. Zilber et al. Biochem. 29:10032-41) isalso useful. The model was used to determine the amino acids in the5C7.29 framework that were close enough to the CDRs to potentiallyinteract with them. To design the humanized light and heavy chain 5C7.29variable regions, the CDRs from the mouse 5C7.29 antibody were graftedinto the framework sequences of the III-3R antibody. At frameworkpositions where the model suggested contact with the CDRs, the aminoacids from the mouse 5C7.29 antibody were chosen to replace the residuesin the III-3R sequence. For humanized 5C7.29, this was done at residues69 and 70 in the light chain and at no residues in the heavy chain.Moreover, at some positions where the amino acid was unusual for humanantibodies at that position, an amino acid representing a consensusamong the relevant human subclass was substituted for the III-3Rframework residue. For humanized 5C7.29, this was done at residues 61,72, 82 and 99 in the light chain and residues 1, 75 and 78 in the heavychain.

The final sequence of the humanized 5C7.29 heavy and light chainvariable region is shown in FIGS. 8A-8B [SEQ ID NOS:5-8]. However, manyof the potential CDR-contact residues are amenable to substitutions ofother amino acids and may still allow the antibody to retain substantialaffinity to the antigens. The following table lists a number ofpositions in the framework where alternative amino acids may be suitable(note LC=light chain, HC=heavy chain):

TABLE 1 Position Humanized 5C7.29 Alternatives LC-1 D Q LC-3 Q V LC-4 ML LC-59 S A LC-69 S D LC-70 Y F HC-49 A S HC-84 N T

Likewise, many of the framework residues not contacting the CDRs in thehumanized 5C7.29 heavy and light chains are also amenable tosubstitutions with amino acids from either the human III-3R antibody, orfrom the corresponding position of other human antibodies, or from themouse 5C7.29 or other mouse antibodies, while still preservingsubstantial affinity and non-immunogenicity of the humanized antibody.The following table lists a number of positions in the framework wherealternative amino acids may be suitable:

TABLE 2 Position Humanized 5C7.29 Alternatives LC-61 F I LC-72 L F LC-82F I, A LC-99 Q G, S HC-1 E Q, D HC-75 S A, P HC-78 T S HC-116 A S

Finally, even certain residues in the CDRs may be substituted with otherresidues while the antibody may retain substantially the same affinityand specificity. Structure-function studies of antibody binding revealthat not all of the CDR amino acids participate equally in specifyingaffinity towards a given antigen (or set of related antigens). Thesestudies enable prediction with some reliability of particular CDRpositions least likely to change substantially the bindingcharacteristics of an antibody. For example, Chothia and co-workersdefine structurally acceptable amino acids in CDR positions (Chothia etal., J. Mol. Biol. 196: 902 (1987); Chothia et al., Nature 342: 877(1989); and Tramontano et al., Proteins: Struct. Funct. Genet. 6: 382(1989)), and many of these are not accessible to solvent (i.e, availableto participate in binding), in the model of 5C7.29. Other workers haveshown that residues 61-66 of CDR H2 may not participate in antigenbinding (Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992);Hsiao et al., Protein Eng. 7:815 (1994)). Surveys of antibody-antigencomplex structures support this notion (Glaser et al., J. Immunol. 149:2606 (1992); Padlan, Mol. Immunol. 31: 169 (1994)). Some of these CDRresidues that may be changed in humanized 5C7.29 and their potentialsubstitutions are listed in the following table:

TABLE 3 Humanized CDR Position 5C7.29 Alternatives L1 29 V I, L 32 M LL2 53 L any 54 A any 55 S T L3 88 Q N, H 89 Q N, H H1 34 M I, V, L H2 61A any 62 D any 63 T any 64 V A, I, L, M, F 65 R K, Q 66 G A, D, T, S

Selection of various combinations of alternative amino acids may be usedto produce versions of humanized 5C7.29 that have varying combinationsof affinity, specificity, non-immunogenicity, ease of manufacture andother desirable properties. The above examples are offered by way ofillustration, not of limitation.

EXAMPLE 7 Construction of Humanized 5C7.29

For the construction of variable region genes for the humanized 5C7.29antibody, nucleotide sequences were selected that encode the proteinsequences of the humanized heavy and light chains, including the signalpeptide, generally utilizing codons found in the mouse sequence. Severaldegenerate codons were changed to create restriction sites or to removeundesirable ones. The nucleotide sequences of the genes also includedsplice donor signals and an XbaI site at each end. The nucleotidesequences and encoded light and heavy chain variable regions of thehumanized 5C7.29 antibody are shown in FIGS. 8A-8B [SEQ ID NOS:5-8].

Each gene was constructed from eight overlapping syntheticoligonucleotides. Assembly and amplification of the genes were carriedout in four steps as shown in FIG. 9: (1) the four pairs ofcomplementary oligonucleotides were annealed and extended with Klenowpolymerase in separate reactions; (2) the resulting four double-strandedDNA fragments were mixed in pairs, denatured, re-annealed and extendedin two separate reactions using Klenow fragment; (3) the resulting twodouble-stranded half-gene fragments were mixed, denatured, re-annealedand extended to create the full length double stranded variable regiongenes; (4) the gene fragments were finally amplified, using Taqpolymerase and two primers that hybridize to the 5′ and the 3′ end ofthe variable region genes and contain XbaI sites for cloning into therespective expression vectors, pVk and pVg4. Reactions were carried outunder conditions well-known in the art.

The pVk vector for light chain expression and the pVg1 vector for heavychain expression have been previously described (see Co et al., J.Immunol. 148: 1149 (1992)). To produce a humanized 5C7.29 antibody ofthe IgG4 isotype, the heavy chain expression vector pVg4 has beenconstructed. To do so, the XbaI-BamHI fragment of pVg1 containing the γ1constant region was replaced with an approximately 2000 bp fragment ofthe human γ4 constant region gene (Ellison and Hood, Proc. Natl. Acad.Sci USA 79:1984 (1982)) that extended from the HindIII side precedingthe CH1 exon of the γ4 gene to 270 bp after the NsiI site following theCH4 exon of the gene, using methods well-known to those skilled in theart, including polymerase chain reaction.

The heavy chain and light chain plasmids were transfected into a mousemyeloma cell line Sp2/0-Ag14 (ATCC CRL 1581). Transfection was byelectroporation using a Gene Pulser apparatus (Bio-Rad) at 360 V and 25uFD capacitance according to the manufacturer's instructions. Beforetransfection, the light chain- and heavy chain-containing plasmids werelinearized using PvuII, extracted with phenol-chloroform, andethanol-precipitated. All transfections were done using 30-50 μg plasmidDNA and about 10⁷ cells in PBS. The cells from each transfection wereplated into 2 to 4 96-well tissue culture plates. After 48 hours,selective medium was applied.

Cells were selected for gpt expression in DMEM+10% FBS+HT mediasupplement (Sigma)+1 μg/ml mycophenolic acid. Antibody-producing cloneswere screened by assaying human antibody production in the culturesupernatant by ELISA. Antibody from the best-producing clones waspurified by passing tissue culture supernatant over a column of proteinA-Sepharose™ CL-4B (Pharmacia). The bound antibodies were eluted with0.2 M glycine-HCl, pH 3.0, and neutralized with 1 M Tris-HCl, pH 8.0.The buffer was exchanged into phosphate buffered saline (PBS) by passingover a PD10 column (Pharmacia), or by dialysis. To obtain cellsproducing higher levels of antibody, the transfected clones may becultured in increasing concentrations of methotrexate.

EXAMPLE 8 Properties of Humanized 5C7.29

To show that humanized 5C7.29 specifically binds to E-selectin andP-selectin, L1-2^(E-selectin) and L1-2^(P-selectin) transfectants wereincubated with humanized 5C7.29 or control antibodies for 1 hour. Afterwashing, cells were incubated in a 1:400 dilution ofphycoerythrin-conjugated anti-human Ig (Biosource, Camarillo, Calif.),washed, then analyzed for fluorescence by flow cytometry (FACS) aspreviously described (Berg et al., Blood 85: 31 (1995)). Humanized5C7.29 reacts with both L1-2^(E-selectin) and L1-2^(P-selectin)transfectants, but not L1-2^(L-selectin) transfectants (FIG. 10)demonstrating that the humanization process did not result in loss ofeither E-selectin or P-selectin binding or gain in the ability to bindL-selectin.

The affinity of the humanized 5C7.29 antibody for E-selectin andP-selectin was separately determined by competition with theradio-iodinated mouse 5C7.29 antibody (FIG. 11). Purified mouse 5C7.29antibody was labeled with Na¹²⁵I (Amersham, Arlington Heights, Ill.)using the lactoperoxidase procedure to 4 mCi/mg of protein.CHO^(E-selectin) cells and L1-2^(P-selectin) cells, which were obtainedby transfecting the E-selectin and P-selectin genes into the respectivehost cells CHO and L1-2 (Gallatin et al., Nature 304:30 (1983)) bymethods well known in the art (see, e.g., Larsen et al., J. Biol. Chem.267: 11104 (1992)), were used as sources for E-selectin and P-selectin.Increasing amounts of competitor antibody (mouse 5C7.29 or humanized5C7.29) were added to 2 ng of radio-iodinated tracer mouse 5C7.29antibody and incubated with 4×10⁵ CHO^(E-selectin) cells orL1-2^(P-selectin) cells in 0.2 ml of binding buffer (PBS with 2% fetalcalf serum, 0.1% sodium azide) for 2 hours at 4° C. with constantshaking. Cells were then washed and centrifuged, and theirradioactivities measured. The ratio of bound and free tracer antibodywere calculated (FIGS. 11A and 11B).

The binding affinities were calculated according to the methods ofBerzofsky and Berkower (J. A. Berzofsky and I. J. Berkower, inFundamental Immunology (ed. W. E. Paul), Raven Press (New York), p. 595(1984)). The humanized 5C7.29 had an affinity of approximately 3×10⁸ M⁻¹for E-selectin, with no significant difference from that of mouse5C7.29, and an affinity of approximately 1.3×10⁸ M⁻¹ for P-selectin,within about 3 to 4-fold of the mouse 5C7.29 antibody. This experimentalso shows directly that humanized 5C7.29 has the ability to competewith the mouse 5C7.29 antibody for binding to both E-selectin andP-selectin. In another experiment, the affinities of mouse and humanized5C7.29 for P-selectin were determined by the method of Scatchard(Berzofsky and Berkower, supra) to be approximately 1.9×10⁸ M⁻¹ and6×10⁸ M⁻¹, respectively, in good agreement with the results of thecompetitive binding experiment.

To show that the humanized 5C7.29 antibody inhibits binding ofE-selectin to a counter-receptor for E-selectin, its ability to inhibitthe binding of HL-60 cells to E-selectin transfectant cells wasdetermined. Assays of the adhesion of HL-60 cells with CHO^(E-selectin)cells were performed as previously described (Berg et al., Blood 85: 31(1995), and supra) in the presence of monoclonal antibodies at theindicated concentrations. FIG. 12 shows that humanized 5C7.29 blocksbinding of HL-60 cells to CHO^(E-selectin) transfectants as well asmouse 5C7.29. For the representative experiment shown, two treatmentsper slide (each treatment in quadruplicate) were analyzed and the meanand standard deviations calculated. An isotype-matched control antibodydid not affect binding.

To show that the humanized 5C7.29 antibody inhibits binding ofP-selectin to a counter-receptor for P-selectin, its ability to inhibitthe binding of HL-60 cells and activated platelets was determined.Assays of the rosetting of activated platelets to the HL-60 cells wereperformed as described (Berg et al., Blood 85:31 (1995) and supra) inthe presence of monoclonal antibodies at the indicated concentrations.FIG. 13 shows that humanized 5C7.29 blocks binding of platelets to HL-60cells as well as mouse 5C7.29. An isotype-matched control antibody hadno affect on binding in this assay. The representative experiment shownwas performed in triplicate and the mean and standard deviationscalculated.

EXAMPLE 9 Epitoge Mapping of 5C7.29

To determine the amino acids of E-selectin involved in the binding of5C7.29 (the epitope), the following procedure was used. DNA encoding thelectin and EGF-like domains of human E-selectin were fused to a geneencoding the human immunoglobulin lambda constant region (C_(λ)), whichserved as a tag. The chimeric DNA was inserted in a plasmid vector,which provided a lac promoter and pelB signal sequence for expressionand secretion of the chimeric (fusion) protein in E. coli. TheE-selectin domains were randomly mutagenized by error-prone polymerasechain reaction (PCR) utilizing AmpliTaq enzyme (Perkin Elmer) and Mn⁺⁺,and the amino acid substitutions were determined by DNA sequencing. E.coli strain TG1ΔrecA was transformed with the wild-type and mutantplasmids, and chimeric proteins were overexpressed by growingtransformed E. coli in 2YT broth. After 8 hours of induction with 1mMIPTG, culture supernatants containing the chimeric proteins werecollected. All operations were performed according to methods well-knownin the art of molecular biology.

Next, 96-well plates were coated with the 5C7.29 antibody (or controlantibodies). After blocking, the plates were incubated with the E. colisupernatants and then with HRP-conjugated anti-human C_(λ) antibodies(Biosource, Camarillo, Calif.). After washing, bound enzyme was detectedwith TMB substrate. Supernatants containing mutant E-selectin-C_(γ)chimeric protein to which 5C7.29 could still bind gave a positivesignal, while supernatants containing mutant E-selectin to which 5C7.29could not bind gave a negative signal. The results are shown in thefollowing table, where the symbol AXB means a mutant in which the Xthamino of E-selectin form the mature N-terminus is changed from thenormal A to mutant B.

TABLE 4 Mutant Reactivity Q21R − R22G − Y23H − Y48H + E92G + N105S +K111E + T119A − A120T −

Because mutating amino acids Q₂₁, R₂₂, Y₂₃, T₁₁₉ and A₁₂₀ in E-selectinprevented binding of antibody 5C7.29, these amino acids must be in theepitope of 5C7.29. The full amino acid sequence of E-selectin is givenin Bevilacqua, supra and in U.S. Pat. No. 5,272,263 (ELAM-1). (Anotheranti-E-selectin antibody was able to bind to these mutants, showing thatthey did not disrupt the overall structure of E-selectin). Other E/Pcross-reacting antibodies that show a different pattern of reactivitywith these E-selectin mutants must have a different epitope inE-selectin. The epitope of 5C7.29 in P-selectin may be determined by asimilar procedure using P-selectin mutants, and may be similarlycompared to the epitope of other E/P cross-reacting antibodies. Theepitopes of 5C7.29 in E-selectin and P-selectin are preferred epitopes,because antibodies such as 5C7.29 that bind to them may have highaffinity and blocking activity.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. All publications and patent documents cited in thisapplication are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

10 384 base pairs nucleic acid single linear cDNA unknown CDS 1..384/note= “mouse 5C7.29 antibody light chain variable region cDNA” 1 ATGGAT TTT CAA GTG CAG ATT TTC AGC TTC CTG CTA ATC AGT GCC TCA 48 Met AspPhe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 GTCATA ATA TCC AGA GGA CAA ATT GTT CTC ACC CAG TCT CCA GCA ATC 96 Val IleIle Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile 20 25 30 ATG TCTGCA TCT CCA GGG GAG AAG GTC ACC ATG ACC TGC AGT GCC AGC 144 Met Ser AlaSer Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser 35 40 45 TCA AGT GTGCCT TAC ATG CAC TGG TAT CAG CAG AAG TCA GGC ACC TCC 192 Ser Ser Val ProTyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser 50 55 60 CCC AAA TTA TGGATT TAT GAC ACA TCC AAT CTG GCT TCT GGA GTC CCT 240 Pro Lys Leu Trp IleTyr Asp Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 GCT CGC TTC AGTGGC AGT GGG TCT GGG ACC TCT TAC TCT CTC ACA ATC 288 Ala Arg Phe Ser GlySer Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 AGC AGC ATG GAG GCTGAA GAT GCT GCC ACT TAT TAC TGC CAG CAG TGG 336 Ser Ser Met Glu Ala GluAsp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 AGT AGT GAC CCA TTCACG TTC GGC TCG GGG ACA AAG TTG GAA ATA AAG 384 Ser Ser Asp Pro Phe ThrPhe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 128 amino acidsamino acid linear protein unknown 2 Met Asp Phe Gln Val Gln Ile Phe SerPhe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Ile Ser Arg Gly Gln IleVal Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser Ala Ser Pro Gly Glu LysVal Thr Met Thr Cys Ser Ala Ser 35 40 45 Ser Ser Val Pro Tyr Met His TrpTyr Gln Gln Lys Ser Gly Thr Ser 50 55 60 Pro Lys Leu Trp Ile Tyr Asp ThrSer Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser GlySer Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Ser Met Glu Ala Glu AspAla Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asp Pro Phe ThrPhe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 405 base pairsnucleic acid single linear cDNA unknown CDS 1..405 /note= “mouse 5C7.29antibody heavy chain variable region cDNA” 3 ATG GAC TCC AGG CTC AAT TTAGTT TTC CTT GTC CTT ATT TTA AAA GGT 48 Met Asp Ser Arg Leu Asn Leu ValPhe Leu Val Leu Ile Leu Lys Gly 1 5 10 15 GTC CAG TGT GAT GTA CGA CTGGTG GAG TCT GGG GGA GGT TTA GTG CAG 96 Val Gln Cys Asp Val Arg Leu ValGlu Ser Gly Gly Gly Leu Val Gln 20 25 30 CCT GGA GGG TCC CGG AAA CTC TCCTGT GCA GCC TCT GGA TTC ACT TTC 144 Pro Gly Gly Ser Arg Lys Leu Ser CysAla Ala Ser Gly Phe Thr Phe 35 40 45 AGT AGC TTT GGA ATG CAC TGG GTT CGTCAG GCT CCT GAT AAG GGG CTG 192 Ser Ser Phe Gly Met His Trp Val Arg GlnAla Pro Asp Lys Gly Leu 50 55 60 GAG TGG GTC GCA TTC ATT AGC AGT GGC AGTAGT ACC ATC TAC TAT GCT 240 Glu Trp Val Ala Phe Ile Ser Ser Gly Ser SerThr Ile Tyr Tyr Ala 65 70 75 80 GAC ACA GTG AGG GGC CGA TTC ACC ATC TCCAGA GAC AGT CCC AAG AAC 288 Asp Thr Val Arg Gly Arg Phe Thr Ile Ser ArgAsp Ser Pro Lys Asn 85 90 95 ACC CTG TTC CTG CAA ATG ACC AGT CTA AGG TCTGAG GAC ACG GCC ATG 336 Thr Leu Phe Leu Gln Met Thr Ser Leu Arg Ser GluAsp Thr Ala Met 100 105 110 TAT TAC TGT GCA AGA CCT TTA CCC CCG TTT GCTTAC TGG GGC CAA GGG 384 Tyr Tyr Cys Ala Arg Pro Leu Pro Pro Phe Ala TyrTrp Gly Gln Gly 115 120 125 ACT TTG GTC ACT GTC TCT GCA 405 Thr Leu ValThr Val Ser Ala 130 135 135 amino acids amino acid linear proteinunknown 4 Met Asp Ser Arg Leu Asn Leu Val Phe Leu Val Leu Ile Leu LysGly 1 5 10 15 Val Gln Cys Asp Val Arg Leu Val Glu Ser Gly Gly Gly LeuVal Gln 20 25 30 Pro Gly Gly Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly PheThr Phe 35 40 45 Ser Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Asp LysGly Leu 50 55 60 Glu Trp Val Ala Phe Ile Ser Ser Gly Ser Ser Thr Ile TyrTyr Ala 65 70 75 80 Asp Thr Val Arg Gly Arg Phe Thr Ile Ser Arg Asp SerPro Lys Asn 85 90 95 Thr Leu Phe Leu Gln Met Thr Ser Leu Arg Ser Glu AspThr Ala Met 100 105 110 Tyr Tyr Cys Ala Arg Pro Leu Pro Pro Phe Ala TyrTrp Gly Gln Gly 115 120 125 Thr Leu Val Thr Val Ser Ala 130 135 384 basepairs nucleic acid single linear DNA unknown CDS 1..384 /note=“humanized 5C7.29 antibody light chain variable region synthetic DNA” 5ATG GAT TTT CAA GTG CAG ATT TTC AGC TTC CTG CTA ATC AGT GCC TCA 48 MetAsp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15GTC ATA ATA TCC AGA GGA GAT ATT CAA ATG ACC CAG TCT CCA TCT AGC 96 ValIle Ile Ser Arg Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 TTATCT GCA TCT GTA GGG GAT AGG GTC ACC ATA ACC TGC AGT GCC AGC 144 Leu SerAla Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser 35 40 45 TCA AGTGTG CCT TAC ATG CAC TGG TAT CAG CAG AAG CCA GGC AAA GCC 192 Ser Ser ValPro Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 50 55 60 CCC AAA TTATTG ATT TAT GAC ACA TCC AAT CTG GCT TCT GGA GTC CCT 240 Pro Lys Leu LeuIle Tyr Asp Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 TCT CGC TTCAGT GGC AGT GGG TCT GGG ACC TCT TAC ACT CTC ACA ATC 288 Ser Arg Phe SerGly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile 85 90 95 AGC AGC CTG CAGCCT GAA GAT TTT GCC ACT TAT TAC TGC CAG CAG TGG 336 Ser Ser Leu Gln ProGlu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 AGT AGT GAC CCATTC ACG TTC GGC CAG GGG ACA AAG GTG GAA ATA AAG 384 Ser Ser Asp Pro PheThr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125 128 amino acidsamino acid linear protein unknown 6 Met Asp Phe Gln Val Gln Ile Phe SerPhe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Ile Ser Arg Gly Asp IleGln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp ArgVal Thr Ile Thr Cys Ser Ala Ser 35 40 45 Ser Ser Val Pro Tyr Met His TrpTyr Gln Gln Lys Pro Gly Lys Ala 50 55 60 Pro Lys Leu Leu Ile Tyr Asp ThrSer Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser GlySer Gly Thr Ser Tyr Thr Leu Thr Ile 85 90 95 Ser Ser Leu Gln Pro Glu AspPhe Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asp Pro Phe ThrPhe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125 405 base pairsnucleic acid single linear DNA unknown CDS 1..405 /note= “humanized5C7.29 antibody heavy chain variable region synthetic DNA” 7 ATG GAC TCCAGG CTC AAT TTA GTT TTC CTT GTC CTT ATT TTA AAA GGT 48 Met Asp Ser ArgLeu Asn Leu Val Phe Leu Val Leu Ile Leu Lys Gly 1 5 10 15 GTC CAG TGTGAA GTA CAA CTG GTG GAG TCT GGG GGA GGT TTA GTG CAG 96 Val Gln Cys GluVal Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 CCT GGA GGG TCCCTT CGT CTC TCC TGT GCA GCC TCT GGA TTC ACT TTC 144 Pro Gly Gly Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 AGT AGC TTT GGA ATGCAC TGG GTT CGT CAG GCT CCT GGT AAG GGG CTG 192 Ser Ser Phe Gly Met HisTrp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 GAG TGG GTC GCA TTC ATTAGC AGT GGC AGT AGT ACC ATC TAC TAT GCT 240 Glu Trp Val Ala Phe Ile SerSer Gly Ser Ser Thr Ile Tyr Tyr Ala 65 70 75 80 GAC ACA GTG AGG GGC CGATTC ACC ATC TCC AGA GAC AAC ACC AAG AAC 288 Asp Thr Val Arg Gly Arg PheThr Ile Ser Arg Asp Asn Thr Lys Asn 85 90 95 ACC CTG TAT CTG CAA ATG AACAGT CTA AGG GCT GAG GAC ACG GCC GTG 336 Thr Leu Tyr Leu Gln Met Asn SerLeu Arg Ala Glu Asp Thr Ala Val 100 105 110 TAT TAC TGT GCA AGA CCT TTACCC CCG TTT GCT TAC TGG GGC CAA GGG 384 Tyr Tyr Cys Ala Arg Pro Leu ProPro Phe Ala Tyr Trp Gly Gln Gly 115 120 125 ACT TTG GTC ACT GTC TCT GCA405 Thr Leu Val Thr Val Ser Ala 130 135 135 amino acids amino acidlinear protein unknown 8 Met Asp Ser Arg Leu Asn Leu Val Phe Leu Val LeuIle Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Val Gln Leu Val Glu Ser GlyGly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala AlaSer Gly Phe Thr Phe 35 40 45 Ser Ser Phe Gly Met His Trp Val Arg Gln AlaPro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Phe Ile Ser Ser Gly Ser SerThr Ile Tyr Tyr Ala 65 70 75 80 Asp Thr Val Arg Gly Arg Phe Thr Ile SerArg Asp Asn Thr Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu ArgAla Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Pro Leu Pro ProPhe Ala Tyr Trp Gly Gln Gly 115 120 125 Thr Leu Val Thr Val Ser Ala 130135 106 amino acids amino acid linear peptide unknown Modified-site/product= “OTHER” /note= “Xaa = Asp or Gln” Modified-site /product=“OTHER” /note= “Xaa = Gln or Val” Modified-site /product= “OTHER” /note=“Xaa = Met or Leu” Modified-site 29 /product= “OTHER” /note= “Xaa = Val,Ile or Leu” Modified-site 32 /product= “OTHER” /note= “Xaa = Met or Leu”Modified-site 53 /product= “OTHER” /note= “Xaa = any amino acid”Modified-site 54 /product= “OTHER” /note= “Xaa = any amino acid”Modified-site 55 /product= “OTHER” /note= “Xaa = Ser or Thr”Modified-site 59 /product= “OTHER” /note= “Xaa = Ser or Ala”Modified-site 61 /product= “OTHER” /note= “Xaa = Phe or Ile”Modified-site 69 /product= “OTHER” /note= “Xaa = Ser or Asp”Modified-site 70 /product= “OTHER” /note= “Xaa = Tyr or Phe”Modified-site 72 /product= “OTHER” /note= “Xaa = Leu or Phe”Modified-site 82 /product= “OTHER” /note= “Xaa = Phe, Ile or Ala”Modified-site 88 /product= “OTHER” /note= “Xaa = Gln, Asn or His”Modified-site 89 /product= “OTHER” /note= “Xaa = Gln, Asn or His”Modified-site 99 /product= “OTHER” /note= “Xaa = Gln, Gly or Ser” 9 XaaIle Xaa Xaa Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Xaa Pro Tyr Xaa 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser Asn Xaa Xaa Xaa Gly Val Pro Xaa Arg Xaa Ser Gly Ser 50 55 60Gly Ser Gly Thr Xaa Xaa Thr Xaa Thr Ile Ser Ser Leu Gln Pro Glu 65 70 7580 Asp Xaa Ala Thr Tyr Tyr Cys Xaa Xaa Trp Ser Ser Asp Pro Phe Thr 85 9095 Phe Gly Xaa Gly Thr Lys Val Glu Ile Lys 100 105 116 amino acids aminoacid linear peptide unknown Modified-site /product= “OTHER” /note= “Xaa= Glu, Gln or Asp” Modified-site 34 /product= “OTHER” /note= “Xaa = Met,Ile, Val or Leu” Modified-site 49 /product= “OTHER” /note= “Xaa = Ala orSer” Modified-site 61 /product= “OTHER” /note= “Xaa = any amino acid”Modified-site 62 /product= “OTHER” /note= “Xaa = any amino acid”Modified-site 63 /product= “OTHER” /note= “Xaa = any amino acid”Modified-site 64 /product= “OTHER” /note= “Xaa = Val, Ala, Ile, Leu, Metor Phe” Modified-site 65 /product= “OTHER” /note= “Xaa = Arg, Lys orGln” Modified-site 66 /product= “OTHER” /note= “Xaa = Gly, Ala, Asp, Thror Ser” Modified-site 75 /product= “OTHER” /note= “Xaa = Ser, Ala orPro” Modified-site 78 /product= “OTHER” /note= “Xaa = Thr or Ser”Modified-site 84 /product= “OTHER” /note= “Xaa = Asn or Thr”Modified-site 116 /product= “OTHER” /note= “Xaa = Ala or Ser” 10 Xaa ValGln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 GlyXaa His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 XaaPhe Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Xaa Xaa Xaa Xaa 50 55 60 XaaXaa Arg Phe Thr Ile Ser Arg Asp Asn Xaa Lys Asn Xaa Leu Tyr 65 70 75 80Leu Gln Met Xaa Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Pro Leu Pro Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105110 Thr Val Ser Xaa 115

What is claimed is:
 1. A recombinant monoclonal antibody comprising afirst amino acid sequence from a first immunoglobulin and a second aminoacid sequence from a second immunoglobulin, and having a binding sitethat specifically binds to P-selectin and to E-selectin, wherein thespecific binding of the antibody to the P-selectin inhibits binding ofthe P-selectin to a counterreceptor of P-selectin; and the specificbinding of the antibody to the E-selectin inhibits binding of theE-selectin to a counterreceptor of E-selectin, and wherein an excess ofantibody reduces the quantity of receptor bound to counterreceptor by atleast 60% as measured in an in vitro competitive binding assay.
 2. Theantibody of claim 1 wherein the counterreceptors are expressed on anHL-60 cell or a neutrophil.
 3. The antibody of claim 1 that competeswith antibody 5C7.29, ATCC accession number CRL 11640, for specificbinding to P-selectin and to E-selectin.
 4. The antibody of claim 1 thatis a mouse antibody.
 5. The antibody of claim 1 comprising a mousevariable region.
 6. The antibody of claim 1 that is a human antibody. 7.The antibody of claim 1 that is a Fab, Fab′, F(ab′)2, Fv fragment, or asingle-chain antibody.
 8. The antibody of claim 1 that does notspecifically bind to L-selectin.
 9. The antibody of claim 1 thatrecognizes an epitope of E-selectin comprising amino acids Q21, R22,Y23, T119, and A120.
 10. The antibody of claim 1 wherein an excess ofthe antibody inhibits binding to counterreceptor by at least 85% asmeasured in an in vitro competitive binding assay.
 11. A pharmaceuticalcomposition comprising the monoclonal antibody of claim
 1. 12. Apurified nucleic acid encoding a light or heavy chain variable region ofthe antibody of claim
 1. 13. A stable cell line comprising: a firstnucleic acid encoding the heavy chain of the antibody of claim 1, thenucleic acid operably linked to a first promoter to allow expression ofthe heavy chain; a second nucleic acid encoding the light chain of theantibody of claim 1, the second nucleic acid operably linked to a secondpromoter to allow expression of the light chain; wherein the stable cellline can produce the antibody of claim
 1. 14. A method of treating aninflammatory disease or condition, selected from adult respiratorydistress syndrome and trauma, comprising administering to a humanpatient a therapeutically effective dose of a pharmaceutical compositioncomprising the antibody of claim
 1. 15. A method of treating aninflammatory disease or condition selected from ischemia-reperfusioninjury after myocardial infarction and stroke comprising administeringto a human patient a therapeutically effective dose of a pharmaceuticalcomposition comprising the antibody of claim
 1. 16. The method of claim15 further comprising the step of administering a therapeuticallyeffective dose of a thrombolytic agent.
 17. A humanzed antibody thatspecifically binds to P-selectin and inhibits the binding of theP-selectin to a counterreceptor of P-selectin; and that specificallybinds to E-selectin and inhibits the binding of the E-selectin to acounterreceptor of E-selectin, said antibody comprising a humanizedlight chain variable region and a humanized heavy chain variable regionwherein the humanized light chain variable region comprises thesequence: DIQMTQSPSS LSASVGDRVT ITCSASSSVP YMHWYQQKPG KAPKLLIYDTSNLASGVPSR FSGSGSGTSY TLTISSLQPE DFATYYCQQW SSDPFTFGQG TKVEIK (SEQ IDNO:6) and the humanized heavy chain variable region comprises thesequence: EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMHWVRQA PGKGLEWVAFISSGSSTIYY ADTVRGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARPL PPFAYWGQGT LVTVSA(SEQ ID NO:8).
 18. The humanized antibody of claim 17 further comprisinghuman light chain and heavy chain constant regions.
 19. The humanizedantibody of claim 17 wherein an excess of the antibody inhibits bindingto counterreceptor by at least 85% as measured in an in vitrocompetitive binding assay.
 20. A pharmaceutical composition comprisingthe monoclonal antibody of claim
 17. 21. A purified nucleic acidencoding a light or heavy chain variable region of the antibody of claim17.
 22. The purified nucleic acid of claim 21 further encoding a humanlight chain or heavy chain constant region.
 23. A stable cell linecomprising: a first nucleic acid encoding the heavy chain of theantibody of claim 17, the nucleic acid operably linked to a firstpromoter to allow expression of the heavy chain; a second nucleic acidencoding the light chain of the antibody of claim 17, the second nucleicacid operably linked to a second promoter to allow expression of thelight chain; wherein the stable cell line can produce the antibody ofclaim
 17. 24. A method of treating an inflammatory disease or condition,selected from adult respiratory distress syndrome and trauma, comprisingadministering to a human patient a therapeutically effective dose of apharmaceutical composition comprising the antibody of claim
 17. 25. Amethod of treating an inflammatory disease or condition selected fromischemia-reperfusion injury after myocardial infarction and strokecomprising administerng to a human patient a therapeutically effectivedose of a pharmaceutical composition comprising the antibody of claim17.
 26. The method of claim 25 further comprising the step ofadministering a therapeutically effective dose of a thrombolytic agent.27. A humanized antibody that specifically binds to P-selectin andinhibits the binding of the P-selectin to a counterreceptor ofP-selectin, and that specifically binds to E-selectin and inhibits thebinding of the E-selecting to a counterrecepctor of E-selectin, saidantibody comprising a humanized light chain variable region and ahumanized heavy chain variable region wherein (1) the humanized lightchain variable region comprises the sequence: X₁IX₂X₃TQSPSS LSASVGDRVTITCSASSSX₁₁P YX₁₂HWYQQKPG KAPKLLIYDT SNX₁₃X₁₄X₁₅GVPX₄R X₇SGSGSGTX₅X₆TX₈TISSLQPE DX₉ATYYCX₁₆X₁₇W SSDPFTFGX₁₀G TKVEIK (SEQ ID NO:9), whereinX₁=D or Q; X₂=Q or V; X₃=M or L; X₄=S or A; X₅=S or D; X₆=Y or F; X₇=For I; X₈=L or F; X₉=F, I or A; X₁₀=Q, G or S; X₁₁=V, I or L; X₁₂=M or L;X₁₃=any amino acid; X₁₄=any amino acid; X₁₅=S or T; X₁₆=Q, N or H; andX₁₇=Q, N or H; and (2) the humanized heavy chain variable regioncomprises the sequence: X₃VQLVESGGG LVQPGGSLRL SCAASGFTFS SFGX₇HWVRQAPGKGLEWVX₁F ISSGSSTIYY X₈X₉X₁₀X₁₁X₁₂X₁₃RFTI SRDNX₄KNX₅LY LQMX₂SLRAEDTAVYYCARPL PPFAYWGQGT LVTVSX₆ (SEQ ID NO:10); wherein, X₁=A or S; X₂=Nor T; X₃=E, Q or D; X₄=S, A or P; X₅=T or S; X₆=A or S; X₇=M, I, V or L;X₈=any amino acid; X₉=any amino acid; X₁₀=amino acid; X₁₁=V, A, I, L, Mor F; X₁₂=R, K or Q; and X₁₃=G, A, D, T or S.
 28. The humanized antibodyof claim 27 where in the humanized light chain variable region, X₁₁=V;X₁₂=M; X₁₃=L; X₁₄=A; X₁₅=S; X₁₆=Q; and X₁₇=Q; and wherein in thehumanized heavy chain variables region, X₇=M; X₈=A; X₉=D; X₁₀=T; X₁₁=V;X₁₂=R; and X₁₃=G.
 29. A pharmaceutical composition comprising themonoclonal antibody of claim
 27. 30. A purified nucleic acid encoding alight or heavy chain variable region of the antibody of claim
 27. 31. Astable cell line comprising; a first nucleic acid encoding the heavychain of the antibody of claim 27, the nucleic acid operably linked to afirst promoter to allow expression of the heavy chain; a second nucleicacid encoding the light chain of the antibody of claim 27, the secondnucleic acid operably linked to a second promoter to allow expression ofthe light chain; wherein the stabe cell line can produce the antibody ofclaim
 27. 32. A metod of treating an inflammatory disease or condition,selected from adult respiratory distress syndrome and trauma, comprisingadministering to a human patient a therapeutically effective dose of apharmaceutical composition comprising the antibody of claim
 27. 33. Amethod of treating an inflammatory disease or condition selected fromischemia-reperfusion injury after myocardial infarction and strokecomprising administering to a human patient a therapeutically effectivedose of a pharmaceutical composition comprising the antibody of claim27.
 34. The method of claim 33 further comprising the step ofadministering a therapeutically effective dose of a thrombolytic agent.35. A method of generating an antibody capable of blocking E-selectinand P-selectin mediated cell adhesion, the method comprising: immunizinga mammal with P-selectin; immunizing the mammal with E-selectin;immortalizing B-cells from the mammal to obtain immortalized cellsproducing antibodies; and selecting an immortalized cell producing anantibody that specifically binds to E-selectin and to P-selectin, andinhibits binding of E-selectin to a counterreceptor of E-selectin andinhibits binding P-selectin to a counterreceptor of P-selectin.
 36. Amonoclonal antibody that specifically binds to E-selectin andP-selectin, said antibody binding to the same epitope of E-selectin asantibody 5C7.29, ATCC accession number CRL
 11640. 37. The monoclonalantibody of claim 36, said antibody further binding to the same epitopeof P-selectin as antibody 5C7.29, ATCC accession number CRL
 11640. 38. Ahumanized antibody that specifically binds to P-selectin and inhibitsthe binding of the P-selectin to a counterreceptor of P-selectin; andthat specifically binds to E-selectin and inhibits the binding of theE-selectin to a counterreceptor of E-selectin, said antibody comprisinga humanized light chain variable region and a humanized heavy chainvariable region wherein (1) the humanized light chain variable regioncomprises a sequence having at least 80% sequence identity to thesequence: DIQMTQSPSS LSASVGDRVT ITCSASSSVP YMHWYQQKPG KAPKLLIYDTSNLASGVPSR FSGSGSGTSY TLTISSLQPE DFATYYCQQW SSDPFTFGQG TKVEIK (SEQ IDNO:6); and (2) the humanized heavy chain variable region comprises asequence having at least 80% sequence identity to the sequence:EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMHWVRQA PGKGLEWVAF ISSGSSTIYYADTVRGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARPL PPFAYWGQGT LVTVSA (SEQ IDNO:8), wherein an excess of antibody reduces the quantity of receptorbound to counterreceptor by at least 60% as measured in an in vitrocompetitive binding assay.
 39. The antibody of claim 38 wheren thesequence identity is at least 90%.
 40. The antibody of claim 38 whereinthe sequence identity is at least 95%.
 41. The antibody of claim 38wherein the counterreceptors are expressed on an HL-60 cell or aneutrophil.
 42. The antibody of claim 38 that competes with antibody5C7.29, ATCC accession number CRL 11640, for specific binding toP-selectin and to E-selectin.
 43. The antibody of claim 38 that is amouse antibody.
 44. The antibody of claim 38 comprising a mouse variableregion.
 45. The antibody of claim 38 that is a human antibody.
 46. Theantibody of claim 38 that is a Fab, Fab′, F(ab′)2, Fv fragment or asingle-chain antibody.
 47. The antibody of claim 38 that does notspecifically bind to L-selectin.
 48. The antibody of claim 38 thatrecognizes an epitope of E-selectin comprising amio acids Q21, R22, Y23,T119, and A120.
 49. The antibody of claim 38 wherein an excess of theantibody inhibits binding to counterreceptor by at least 85% as measuredin an in vitro competitive binding assay.
 50. A pharmaceuticalcomposition comprising the monoclonal antibody of claim
 38. 51. Apurified nucleic acid segment encoding a light or heavy chain variableregion of the antibody of claim
 38. 52. A stable cell line comprising: anucleic acid segment encoding the heavy chain of the antibody of claim38, the segment operably linked to a first promoter to allow expressionof the heavy chain; a second nucleic acid segment encoding the lightchain of the antibody of claim 38, the second segment operably linked toa second promoter to allow expression of the light chain; wherein thestable cell line can produce the antibody of claim
 38. 53. A method oftreating an inflammatory disease or condition, selected from adultrespiratory distress syndrome and trauma, comprising ministering to ahuman patient a therapeutically effective dose of a pharmaceuticalcomposition comprising the antibody of claim
 38. 54. A method oftreating an inflammatory disease or condition selected fromischemia-reperfusion injury after myocardial infarction and stokecomprising administering to a human patient a therapeutically effectivedose of a pharmaceutical composition comprising the antibody of claim38.
 55. The method of claim 54 further comprising the step ofadministering a therapeutically effective dose of a thrombolytic agent.